Method for the crystallization of human serum albumin

ABSTRACT

The present invention relates to the purification and production of human albumin from various sources through crystallization and repeated crystallization. Basic features of the invented process include providing specific reaction conditions and precipitating reagents to maximize albumin crystallization. Solubility diagrams are utilized as the basis for process control of the invented method. The current invention specifically controls phosphate concentration, pH and temperature to precisely guide crystallization kinetics and crystal yield.

FIELD OF THE INVENTION

The present invention relates to methods for provide a highly reliablemethod and commercially viable method of crystallizing human albumin.More specifically, the current invention provides a method to producecrystalline human albumin purified from various albumin sources,specifically including from transgenic animals or other recombinantsources.

BACKGROUND OF THE INVENTION

The present invention is directed to an improved method of crystallizinghuman serum albumin (“hSA”)(herein, hSA will be used interchangeablywith the term human serum albumin). This process is preferably done toenhance purification procedures for recombinant hSA that can then beutilized in therapeutic applications or as an excipient inpharmaceutical preparations. With regard to pharmaceutical preparationshuman albumin as purified herein can be used as a therapeutic agent oras an excipient. In either case suitable formulations can be found inREMINGTON'S PHARMACEUTICAL SCIENCES (16th and 18th Eds., MackPublishing, Easton, Pa. (1980 and 1990)), and in INTRODUCTION TOPHARMACEUTICAL DOSAGE FORMS (4th Edition, Lea & Febiger, Philadelphia(1985)), each of which is incorporated herein by reference.

For therapeutic applications of hSA the objective of albuminadministration is primarily to maintain circulating plasma volume bymaintaining the plasma colloid oncotic pressure, and to treat otherwiseresistant severe edema by making intracavital and interstitial fluidsmove into the blood vessels.

Albumin products are used to achieve transient improvement of thecondition by replenishing albumin in pathological conditionsattributable to acute hypoproteinemia, and pathological conditionsresulting from chronic hypoproteinemia which is resistant to othermethods of treatment.

Albumin was the first natural colloid composition for clinical use as ablood volume expander, and it is the standard colloidal agent forcomparison with other colloid products. Some of the specific medicalindications in which albumin may be used to increase intravascularoncotic pressure and thereby expand intravascular volume in patientsinclude: hypovolemic shock; severe burn injury; adult respiratorydistress syndrome (ARDS); ascites; liver failure; pancreatitis and inpatients undergoing cardiopulmonary bypass. (Cochrane et al., 1998).Albumin may also be used to treat neonatal hyperbilirubinemia,hypoproteinemia, and nephrotic syndrome. (Vermeulen et al., 1995).

The albumin portion of human blood serves three primary physiologicroles: (1) maintenance of plasma colloid osmotic pressure, (2) transportand sequestration of bilirubin, and (3) transport of fatty acids andother intermediate metabolites such as hormones and enzymes. (Peters, Tet. al.,). Because albumin accounts for approximately 80% of the oncoticpressure of plasma, a 50% reduction in serum albumin concentrationconsequently produces a 66% decrease in colloid oncotic pressure.(Rainey T. G., et al., 1994). In critically ill patients, risk of deathis inversely related to serum albumin concentration. (Cochrane et al.,1998). Goldwaser and Feldman estimate that for each 2.5 g/dL decrease inserum albumin concentration, there is a 24%-56% increase in the risk ofdeath. (Goldwaser et al., 1997). This estimate was made after adjustingfor other co-variants (e.g., renal function, serum trans-aminase, lacticacidosis), and it strongly indicates that albumin infusion may have adirect cytoprotective effect. (Cochrane et al., 1998).

Given the above, it is clear that hSA is perhaps the best known of allthe plasma proteins judging both by the amount of scientific literatureavailable describing it as well as through the number of industrial usesit enjoys. However, this abundant amount of knowledge is focusedprimarily on its physiology and the clinical use of albumin, not themethodology used to purify it or sourcing the molecule from anythingother than plasma fractionation. The best-known and still widely usedpurification methods were developed by Cohn and co-workers 60 years ago(Cohn E. J. et al., 1947). The Cohn plasma fractionation method isprimarily used to produce purified plasma products for a wide variety ofclinical uses. Cohn also developed a widely used crystallization processwhich utilizes principles similar to those well-known from plasmafractionation processes for use with human serum albumin. However, theprocess has significant inefficiencies and often does not provide anadequate supply of highly purified hSA.

Effect of pH

The effect of pH is one of the major factors in protein crystallization.Usually protein crystals have a well-defined minimum point of solubilityat a specific pH. In the general literature of protein crystallization,it is most often the case that this minimum solubility is at theisoelectric point of the target protein. However, hSA is highly solubleat its isoelectric point, across a wide range of ionic strengths. Thus,the crystallization properties of albumin are much more complex thanthose of many other proteins making reliable crystallization and/orpurification problematic.

Albumin has a varying isoelectric point depending on the chemicaltreatment that it has received. With a full complement of six boundfatty acids hSA's pI is normally 4.6, however, when fully de-fatted itspI may be as high as 5.6. Therefore, the crystallization properties ofhSA vary as between its “native” and de-fatted states and the reportedoptimum pH for the crystallization of hSA itself varies substantially inthe literature from a low of pH 4.6 to a high of pH 8.0 and may behighly dependent upon the molecular state of hSA in a batch-by-batchbasis. Thus, the wide range of pH that is considered to be optimal forthe crystallization of hSA present in the literature is confusing andapparently relies on various precipitating reagents, each of which isutilized having a variable concentration and which may be optimal foronly one of the possible molecular states of hSA.

For example, with hSA at low ionic strength, like that expected in theCohn alcohol process, crystallization proceeds optimally at pH 4.9-5.3which is close to the isoelectric point of native albumin. In conditionswith a higher ionic strength and when strongly buffered the optimal pHfor crystallization by PEG solutions is 7.4. In sum, the reported pHeffects for optimal crystallization of hSA are dependent on the reagentcomposition in such a seemingly irregular way that solid conclusionscannot be made by reference to the prior art and prior art methodology.In fact, given the status of the teachings of the prior art, every newreagent and technique must be laboriously optimized according to aspecific pH or other single variable to be kept constant whilecrystallization conditions are worked out.

Effect of Precipitating Reagents

It should be noted that hSA has a very high solubility in varying saltconcentrations. It can be precipitated or crystallized at low ionicstrength with added ethanol (Cohn process; Cohn E. J. et al., 1947) orother solvents. Alternatively, salting out with very high saltconcentration is possible, and the early literature mostly used ammoniumsulfate or ethanol is described. In the more recent literature PEGsolutions of various molecular weights have be utilized widely. However,the reagents present in the literature are unacceptable for the clinicaluse of the resulting hSA because of the remaining contaminants. Of theprior art precipitating reagents only ethanol and ammonium sulfate areuseful in the production of has. However, they both have significantpractical problems. Crystallization with ethanol requires the additionof toxic organic modifiers such as benzene or heavy metals. Ammoniumsulfate is not a suitable salt for final albumin formulation, and wouldthus need to be removed.

Effect of Specific Reagents

In addition to its other characteristics albumin has an extraordinarycapacity to combine and attach to a wide variety of smaller moleculesand ions. The association of various long chain alcohols and fatty acidswith hSA strongly affect the crystallization profile of molecular hSAand again act to make the production of clinical grade human serumalbumin highly variable and unpredictable. Examples of reagents capableof significantly effecting the crystallization profile of hSA include:decanol, palmitic acid and caprylic acid.

Effect of Temperature

In should also be noted that prior art attempts to crystallize hSA inethanol solutions have typically been made at low temperatures in therange of 0-10° C. High salt and PEG procedures are often made at a widertemperature range of 4-20° C. In these prior art efforts it is not clearwhat the effect of temperature really is on albumin precipitation. Inethanol the crystal solubility is seemingly lower at low temperature.The effect of temperature is not clearly described in the PEG and saltmethods found in the prior art. For production efficiency and commercialviable processes temperature is one of the major factors. Overall, thesignificance of temperature is not explained or disclosed by the priorart.

Kinetics and Seeding

According to the prior art, the time period needed for thecrystallization of albumin in a given reaction to be complete can takeup to several days. However, of the prior art methods those employingethanol may be the most rapid, requiring only 12 to 24 hours to initiatecrystallization. Also according to prior art methods the actualcrystallization of albumin may not be possible at all without additivesincluding seeding a reaction mixture with crystals formed from a priorreaction. In addition, the methods of crystallization relying on PEG,may or may not utilize this type of additive. It should be noted thatseeding does speed up the crystal growth significantly, though given theconfusion in the art generally there are no references that can beutilized which give consistently reliable results or generate a highyield of crystal. TABLE 1 Example List of Crystallizing Reagents andConditions Found in the Literature. buffers temp. Precipitants Additives0.05-0.1 M pH ° C. References (NH₄)₂SO₄ 50% Decanol phosphate 4.6-7.74-6 Haupt, H. and Heide, K. Klin. Na₂SO₄ 15-20% acetate 5.0-6.8Wochenschr. (1967), 45(14), K-phosphate 2.2M 5.9 20  pp. 726-729.Na-phosphate 3M 6.8 4-6 PEG 180-800 K-phosphate 4.6-7.2 4 (1) Carter, D.C. EP 0 357 40% Na-acetate 6.8 857 A1 and PEG 400 Na-citrate, 5.5-7.2(2) Carter, D. C. et.al. 40% Tris Science 244 (4909) (1989) p. 1195 PEG3350 long chain phosphates 7.5 (1) Carter, D. C. 17.5% fatty acidsNa-acetate 4.6-8.0 U.S. 5.585.466 and Na-citrate, 7.0-7.5 22  (2)Carter, D. C. et.al. Eur. J. Tris Biochem. 226(3)(1994) p. 1049 PEG 3350K-phosphate 7   4 Bhattacharya et al J. Biol. 28-30% Chem. 275(49)(2000)p. 38731 PEG 400, 4000 K-phosphate 5.0-5.5 15-20 Sugio, S. et.al. Prot.Eng. 20-38% 7.0-8.0 12(6) (1999) p. 439 (NH₄)₂SO₄ Decanol K-phosphate6   4 Rao, S. et.al. J. Biol. Chem. 45% saturated 251(10) (1976) p. 3191MPD Decanol 5.2 1 McClure, R. J. et.al. J. Mol. 0.1% Biol. 83(4) (1974)p. 551 Ethanol 5.2 2 Low, B. W. J. Am. Chem. Soc. 74(1952) p. 4830(NH₄)₂SO₄ Decanol 6.8 1 Low, B. W. and Weichel, E. J. 54% saturated.0.2% J. Am. Chem. Soc. 73(1951) p. 3911 Methanol Numerous acetate4.4-6.5 −5-+5 Lewin, J. J. Am. Chem. Soc. Ethanol 5-30% compounds4.9-5.1 73 (1951) p. 3906 Acetone heavy metals Ethanol, mole CHCl₃ 4.9 −5-+10 Cohn, E. J. et.al. J. Am. fractions 0.02-0.163 Decanol, 5.3Chem. Soc. 69 (1947) p. 1753 benzene, Ethanol 15% Decanol 5.2 <0  Hughes, W. L. J. Am. Chem. HgCl₃ Soc. 69 (1947) p. 1836Prior Art Methodology with Mineral Salts

The prior art (Haupt and Heide (1967)) provided methods to crystallizehuman serum albumin with various mineral salts including: 50% saturated(NH₄)₂SO₄; 15-20% Na₂SO₄; 2.2M K-phosphate pH 6.8 and 3M Na-phosphate pH5.0. Decanol was found to be a necessary crystallization aid in theseprior art methods. Other fatty alcohols with more than five carbon atomsin their molecular backbone were found to be useful also. However, thecrystallization conditions and procedures were very sparingly described.No material balances were presented. On the basis of the data availablefrom this citation it is not possible to perform crystallization ofalbumin in a sufficiently controlled or reliable way.

A review of the prior art literature indicates that while there areseveral methods of crystallization proposed the relevant citations donot teach a process that is efficient at an industrial or commercialscale, teach a method that is unavailable for use in the production of atherapeutic product or excipient, or provide a process only useful inthe production of single crystals useful only in x-ray diffractionstudies. Thus, the limitations of the prior art prevent the developmentof the extensive knowledge of crystallizing conditions necessary in thedesign of a large-scale crystallization processes for hSA in atherapeutic, pharmaceutical excipient, or medical adjuvant role.Moreover, the prior art does not provide teachings that provide for theuse from sources other than human plasma. Therefore, a need exists tounderstand the physical and chemical conditions which produce crystalsof albumin reliably and on an commercial scale from a variety ofsources.

SUMMARY OF THE INVENTION

The present invention provides improved methods for producingcrystalline human albumin. The method is suitable for various albuminsources including: human plasma, and from recombinant albumin sourcessuch as cultured mammalian cells, the milk or other bodily fluids oftransgenic mammals, transgenic plants, transgenic avians, recombinantbacterial cell cultures, recombinant yeast cell cultures or recombinantinsect cell cultures. That is, it is useful for the production ofcrystallized and pharmaceutically grade hSA regardless of the feedstreamfrom which it comes. The method has a high purification power so thatcrystalline albumin can be effectively separated from other proteins,bacteria, fatty acids or other molecular species present in a particularstarting material or feedstream. In an additional embodiment of thecurrent invention, albumin can be dissolved and re-crystallized byheating and cooling cycle in the crystallizing medium. This,re-crystallization procedure can be repeated unlimited number of timesaccording the needs of the user of the current invention. Purity ofalbumin is regularly improved in the re-crystallization procedure.

The methods of the current invention also provide precise combinationsof reagents and conditions that allow the optimization of the productionof crystalline human albumin. In these methods important the processparameters such as pH and temperature are precisely manipulated. Anadditional embodiment of the current invention provides optimalconcentrations of precipitating agents of sodium or potassium phosphatesand/or caprylic acid or caprylate salts.

The process of the current invention is based on certain key factorsinfluencing the crystallization of human albumin. Preferably, theprocess of the current invention optimizes the following variables in aspecific manner so as to optimize the crystallization protocolparameters as follows:

-   -   1. changing the phosphate salt concentration in planned steps;    -   2. varying the temperature of the reaction mixture in planned        steps wherein heating and cooling procedures are applied        successively;    -   3. controlling the pH or varying the pH of the reaction mixture        planned steps manner;    -   4. wherein the application of these specific steps allows the        purification and crystallization of human albumin from a given        feedstream.

Other features and advantages of this invention will become apparent inthe following detailed description of preferred embodiments of thisinvention, taken with reference to the accompanying drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a process flow diagram for crystallization of recombinanthuman serum albumin.

FIG. 2 shows albumin solubility at different temperatures at pH 6.2,with exponential trendlines.

FIG. 3 shows albumin solubility at different temperatures at pH 6.2,with linear trendlines

FIG. 4 shows pH Solubility of albumin crystals at 15° C.

FIG. 5 provides a solubility study of washed albumin crystals indifferent phosphate solution at a pH of 6.2.

FIG. 6 provides a solubility study of washed albumin crystals in a 2.63Mphosphate solution with varying caprylate concentration.

FIG. 7 shows a heat precipitation study of hSA crystal slurry in 2.7 Mphosphate pH 6.2.

FIG. 8 shows a process flow diagram for preparative crystallization ofalbumin.

FIG. 9 shows a process flow diagram for crystallization of recombinanthuman serum albumin.

FIG. 10 shows a process flow diagram for the re-crystallization ofrecombinant human serum albumin.

FIG. 11 shows crystallized human albumin in a solution mixture that is2.3 Molar Na—K-Phosphate with a pH of 6.2; Caprylate 1.4 mg/ml; hSA 80mg/ml crystallized at 4° C. overnight, with two hours at roomtemperature (RT) in an air tight chamber.

FIG. 12 shows crystallized human albumin. shows crystallized humanalbumin in a solution mixture that is 2.5 Molar Na—K-Phosphate with a pHof 6.2; saturated with Caprylate; hSA 46.5 mg/ml crystallized at 4° C.overnight.

FIG. 13 shows crystallized human albumin in a solution mixture that is2.3 Molar Na—K-Phosphate with a pH of 6.2; Caprylate 1.4 mg/ml; hSA 80mg/ml crystallized at 4° C. overnight, with 0 hours at room temperature(RT) in an air tight chamber.

FIG. 14 shows, albumin crystals of the invention along with moreamorphous precipitate. The typical crystal size is approximately 0.1×0.4mm. The precipitate disappears over time becomes crystalline. Thecrystals were prepared at 4° C. in 2.7 Molar ammonium sulfate containing0.08% decanol; 0.05 M Na—K phosphate at pH 7.4. Crystallized at 4° C.hSA 58.7 mg/ml.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following abbreviations have designated meanings in thespecification:

Abbreviation Key:

-   -   pH A term used to describe the hydrogen-ion activity of a        chemical or compound according to well-known scientific        parameters.    -   PEG An abbreviation for polyethylene glycol

Explanation of Terms:

-   -   Colloids—Refers to large molecules that do not pass readily        across capillary walls. These compounds exert an oncotic (i.e.,        they attract fluid) load and are usually administered to restore        intravascular volume and improve tissue perfusion.    -   Diafiltration—An operation incorporating ultrafiltration        membranes to efficiently remove salts or other small molecules        from a macromolecular solution. The purpose is to remove small        molecules from albumim in soltuon and adjust the buffer for the        next procedure.    -   Tissue Perfusion—The amount of blood flow to tissue.    -   Feedstream—The raw material or raw solution provided for a        process or method and containing a protein of interest.

The methods of the current invention for the crystallization of hSAprovide a highly desirable method to separate and purify albumin from afeedstream containing a variety of other protein components. Crystalsare the most pure form of protein, once precipitated crystals havesignificantly better mechanical handling properties than amorphousprecipitates and can be separated by a variety of methods known in thefield. For example, crystals can be separated and washed efficiently onindustrial filters. Crystallization is the most used final purificationmethod of fine chemicals and pharmaceuticals.

According to a preferred embodiment of the current invention albumin iscrystallized with a mixture of sodium and potassium phosphates.Crystallization is optimized by using the invented process conditionsand methods in a systematic manner. Albumin may precipitate as amorphousphase, liquid droplets or gel if the conditions are not adjustedoptimally. Amorphous precipitate is very difficult to handle, it can notbe separated and washed efficiently on filters. Amorphous phase does notreadily convert to crystals. Crystals production is optimized when theprocess conditions are adjusted according to this invention. Variousembodiments of the current invention are provided below.

1. Phosphate Salts:

Mixtures of sodium and potassium phosphates are preferably used,although crystallization can be made either with sodium phosphate aloneor potassium phosphate alone. Phosphate salts NaH₂PO₄ and K₂HPO₄ arepreferred since they can be dissolved in concentrates of up to 4 molaraqueous solutions. These 4 M phosphates can be mixed in all proportionsto make 4 M crystallization buffers with various desirable pH values.

Preferably, albumin is crystallized in relatively high phosphateconcentration, typically around 2.7 M or higher. The significance ofphosphate concentration is most clearly revealed in the FIGS. 1 and 2,which describe the solubility of albumin crystals in phosphates withdifferent molarities. The semi-logarithmic plot of FIG. 2 shows thatcrystal solubility is systematically lowered by increasing phosphatemolarities. Albumin yield can be adjusted in the process to a desirablelevel by using increasing molar concentrations of phosphates. Upperlimits for usable phosphate concentrations are set by the solubility ofphosphate salts. Phosphate solubility is lowered by lowering temperatureand increasing albumin concentration. In one embodiment of the currentprocess phosphate solubility is significantly lowered by reducing thetemperature of the reagent to below 110° C. By lowering the temperatureof the reagent and increasing the albumin concentration the availablewater concentration is also being lowered.

According to a preferred embodiment of the current invention, most ofthe impurities precipitate effectively in starting material in the rangeof phosphate concentration 2.0-2.7 M, where albumin does not readilycrystallize at room temperature (25-30° C.). The impurities are removedby being filtered (after adjusting phosphate concentration 2.0-2.7 M).Thereafter the filtrate is refrigerated to 10° C. where albumin iscrystallized. Furthermore, phosphate concentration can be increased(utilizing the information of FIGS. 1 and 2) in order to increasealbumin yield in the crystals, see also Table 2 and FIG. 4.

2. Significance of pH:

According to the current invention the pH of the crystallizing batch isfully controlled with the phosphate mixture. Examples of the mixtureswith various pH values and the resulting effect on the albumin crystalsare presented in the Table 1. The effect of pH on the albumin crystalsolubility is presented graphically in the FIG. 2, see also Table 2.Albumin crystal solubility is lowered by lowering pH from pH 5.6 toapproximately 6.6. The crystal solubility remains at very low level upto at least pH 7.4. Crystals are completely dissolved below pH 5.5.

The pH has a specific effect on the crystallization kinetics, thus thehigher pH range 6.3-7.4 can not be used in a simple way. In the higherpH range, albumin precipitates as amorphous (liquid droplet) phase ifsuch pH is adjusted right in the beginning of the process. Thus thecrystallization process is preferably made initially with the phosphatepH 6.2 (see table 1 for mixing recipe of phosphates and FIGS. 2 and 3).Later on when albumin is mostly crystallized, phosphate concentration isincreased and pH adjusted to higher value in order to increase thecrystal yield. Higher pH is also used advantageously when washingcrystals, since the loss of albumin is reduced.

3. Effect of Temperature

The solubility of albumin crystals at different temperatures andphosphate concentrations is presented graphically in the FIG. 3. Albumincrystal solubility is increasing at higher temperatures. Lowestsolubility is found around 0° C. However, the preferred crystallizingtemperature is around 110° C., since at lower temperature phosphate maycrystallize and bring the process out of control. The example proceduresaround 0° C. were possible to be performed, since phosphatecrystallization is relatively slow.

The crystallization kinetics of albumin is very slow at roomtemperature. Thus the phosphate concentration can be adjusted to 2.6-2.7at room temperature without precipitating or crystallizing albumin.However, impurities are readily precipitated at room temperature. Afterbeing filtered, albumin solution is refrigerated to preferably 110° C.The solution is stirred and albumin is crystallized.

Albumin can be re-crystallized by utilizing a heating cycle. Thecrystals are dissolved by heating to 45° C. Albumin is crystallizedagain after refrigeration to 10° C. Re-crystallization can be used toimprove the crystal purity. After each crystallization, the motherliquor can be removed by being filtered. Crystals can be washed on thefilter with cold 3 M phosphate. Re-crystallization cycle can be repeatedunlimited number of times. Recrystallization can be used to improvealbumin purity. For convenience please see Tables 7 and 8 and FIG. 7.

Albumin is relatively heat stable. It can be heated up to 65-70° C. forprolonged periods. Most other proteins denature and precipitate at suchhigh temperatures. Thus heating treatment at 65-70° C. can be used topurify albumin solution prior to crystallization. The highest tolerabletemperature is related to the composition and pH of albumin solution. Inhigh phosphate concentration pH 6.2 the highest temperature is 65° C. Inlow salt medium and pH 5.4 heating at 70° C. for 2-3 hours is possible.Examples of the effect of heat treatment are presented in the Tables 2and 3 and FIG. 6.

4. Caprylate

Albumin needs to be saturated with caprylate to be able to crystallize.Caprylate has also a stabilizing effect on the albumin, specially onheat stability. Other long chain fatty acids and long chain alcohols arealternatively useful. Decanol is very effective and well known in priorart. Caprylate has dual effect depending of how it is used. It isbeneficial when it is used only to saturate the binding sites ofalbumin. However, excess of caprylate will dissolve crystals and reducealbumin yield.

The effect of caprylate is well revealed in the FIG. 4. Addition ofcaprylate to the washed crystals clearly increased crystal solubility.Crystal solubility was rapidly increasing when caprylate was increasingup to 10 mM. The solubility increment was smaller but still significantwhen caprylate was increasing from 10 mM to 20 mM. At phosphateconcentration 2.63 M and temperature 10° C., albumin solubilityincreased from 9 mg/ml in 10 mM caprylate to 21 mg/ml in 10 mMcaprylate. At higher phosphate concentration 2.82 M and temperature 10°C., albumin solubility increased from 2 mg/ml in 0 mM caprylate to 12mg/ml in 10 mM caprylate.

Dissolving effect of caprylate is so significant that concentration offree caprylate should be well controlled and maintained as low aspossible in the crystallizing step. Small level of free caprylate, orderof 1-2 mM may be acceptable when conditions are otherwise such thatalbumin crystal solubility is very low. Please see FIGS. 2 through 7.

5. Albumin: Concentration, Purity

In experimental solutions albumin concentration in the starting solutionis set to a level that is higher than the solubility of crystals in theconditions utilized. According to the current invention, when workingwith a feedstream sourced from a transgenic animal or cell cultureinitial clarification steps are typically used to provide a solution inwith the concentration of albumin and other chemical parameters areadjusted or manipulated such that it is also higher than the solubilityof hSA crystals. In both of these situations albumin recovery can beestimated by using the solubility information in the phase diagrams(FIGS. 1-4). According to the current invention concentration levels ofalbumin in feedstream solutions are typically in the range of 15-300grams of albumin in one liter of crystallizing batch. In feedstreamsfrom biological sources and usable for the commercial or industrialproduction of hSA these same ranges are encountered.

Moreover, albumin need not be pure in the crystallizing processaccording to the preferred embodiments of the current invention. Theprocesses developed—and provided by the current invention can beutilized to crystallize out albumin in source material wherein the levelof purity is approximately 10%, that is, where albumin constitutes only10% of the total protein of a given solution. For example, with hSAsourced from either transgenic sources or cell cultures most of theimpurities remaining after clarification procedures can be removed afterprecipitation with the first addition of phosphate up to 2.6 Mconcentration level. It should be noted that transgenic sources,typcially milk, but also including other bodily fluids such as blood orurine may contain hSA as a consequence of the insertion of DNAconstructs designed to cause the stable expression of hSA (or otherprotein of interest) in those bodily fluids or tissues. After theprecipitation and being filtered, the filtrate is further concentratedby ultrafiltration in order to increase albumin concentration level.Thereafter the concentrated albumin solution is refrigerated andcrystallized according to the current invention.

EXAMPLE 1 A Preferred Crystallization Process

This process description is made for the crystallization of a purifiedalbumin solution and for that purposes describes the use of only aphosphate solution only. However, according to the current inventionthis method can be used on impure or only partially purified startingmaterial, as may be found from transgenic or cell culture feedstreams,with the addition of additional steps provided herein. Variations of theinventive method, for example utilizing starting solutions withsignificant impure material, are presented below.

Step 1. Precipitation

Phosphate stock solution, containing 2.8 moles of NaH₂PO₄ and 1.2 molesof K₂HPO₄ dissolved in water and filled to 1.0 liter, was used incrystallization. This 4 M phosphate solution had pH 6.2 when measuredafter dilution to 0.5 M. Thereafter, 200 ml of a purified albuminsolution was precipitated by adding 371 ml of 4.0M phosphate stocksolution at a pH of 6.2. On the basis of the added volume, the phosphateconcentration was 2.6 M. The solution was allowed to precipitate at roomtemperature for 4-18 hours, for this variation of the current inventionthe time period of interest is short relative to prior art methods, inall cases less than 24 hours. The amount of the produced precipitate isdirectly related to the amount of impurities in the albumin solution.

Step 2. Filtration

Thereafter, the precipitated hSA was filtered through glass fiber orcellulose fiber paper having approximately 1 μm pore size. The filteredsolution was then used in a preferred crystallization procedureaccording to the invention.

Step 3. Crystallization

Crystallization was performed in 10° C. thermostat incubator. The batchwas stirred slowly (approximately 70 rpm) with a top driven propeller.Phosphate concentration was increased gradually from 2.6 M to 3 M byadding 229 ml of 4M phosphate which was at a pH of 6.2. Thecrystallization batch was continued for a period of 4 days beforeharvesting and washing.

Step 4. Harvesting and Washing of Crystals

According to a preferred embodiment of the current invention hSAcrystals were harvested by filtering the batch with glass fiber paper (1μm pore size, 142 mm diameter). After being filtered, crystals were thenwashed with about 80 ml of cold 3 M phosphate pH 6.2. Crystals were thensuspended in about 150 ml of cold 3 M phosphate pH 6.2.

With regard to Table 2 below and the preferred crystallization processesof the current invention, it is preferably if the mixing volume ratiosare kept fixed. Preferably, the pH should not be adjusted after makingthe buffer mixture. For this embodiment of the invention the pH valuesare approximate, since the value will change with the alteration inphosphate concentration. The buffers of the current invention were usedin the study for effect of pH on crystal solubility. As seen below inTable 2, Buffer No: 10 provides conditions that are consistentlyoptimized for crystal development and the conditions provided for thisbuffer are the preferred conditions for the standard buffer of thecurrent invention. (See also, FIGS. 11-13). TABLE 2 Mixing Table For 4 MPhosphate Buffers mixing volumes of 4 M A and B Buffer Diluted A 4M B 4MBehavior of hSA crystals No: PH NaH₂PO₄ K₂HPO₄ in 2.6-2.8 M buffers 15.3 95 5 hSA dissolved 2 5.4 94 6 hSA dissolved 3 5.5 92 8 hSA dissolved4 5.6 90 10 hSA Crystallized 5 5.7 87 13 hSA Crystallized 6 5.8 84 16hSA Crystallized 7 5.9 80 20 hSA Crystallized 8 6.0 77 23 hSACrystallized 9 6.1 74 26 hSA Crystallized 10 6.2 70 30 hSA Crystallized11 6.3 65 35 hSA Crystallized 12 6.4 60 40 hSA Crystallized 13 6.5 55 45hSA crystals are stable 14 6.6 50 50 hSA crystals are stable 15 6.7 4357 hSA crystals are stable 16 6.8 39 61 hSA crystals are stable 17 6.935 65 hSA crystals are stable 18 7.0 30 70 hSA crystals are stable 197.1 27 73 hSA crystals are stable 20 7.2 24 76 hSA crystals are stable21 7.3 21 79 hSA crystals are stable 22 7.4 18 82 hSA crystals arestablePHOSPHATE BUFFERSpH values are for diluted buffers in the range of 0.1-0.4 M.The buffer No: 11. pH 6.3 is not much influenced by dilution.The buffers No: 1-10 have lower pH when M is higher.Buffers No: 12-30 have higher pH when molarities are higher.

It should also be noted that in FIG. 6 of the current disclosureprovides a profile of albumin solubility in 2.63 M phosphate as afunction of caprylate concentration and temperature: open circles (∘)mark samples incubated at 10° C. and black squares (▪) mark samplesincubated at 5° C.

EXAMPLE 2 Crystallization of Albumin with the Microdiffusion Method

Crystallization examples from 48 samples were prepared according to ahanging drop microdiffusion method known in the art. The sample solutioncontained purified 198 mg/ml of albumin and 3.2 mg/ml sodium caprylate.The liquid solution of albumin was prepared by mixing 3 μl of albuminsolution with 3 μl of 1.8-2.3 M phosphate. The drops were allowed toequilibrate in the closed microdiffusion wells and refrigerated at 5° C.Crystals were produced in less than 24 hours according to thisembodiment of the the current invention. According to the currentinvention, feedstreams from other source material, especially transgenicand cell culture sources, can also be utilized in conjunction with amicrodiffusion hanging drop method.

The crystals were observed with microscope and photographed with digitalcamera. Information regarding the development of the crystals areprovided on tables 19 and 20. These examples show that caprylatesaturated albumin is crystallized in 1.8 M through 2.3 M phosphates in asharply defined pH range, that is, from 5.0-6.4. The most preferred pHfor crystal formation was at pH 6.2, as seen below in Table 3. The rangeof experimental conditions provided effectively covers the range ofconcentrations and solution parameters that have been seen in alternatefeedstream sources, see Table 4. TABLE 3 Sample: Mix 90 μl hSAconcentrate 210 mg/ml and 10 μl caprylate solution (32 mg/ml pH 7.3.).Final caprylate concentration is 3.2 mg/ml. Drop: 3 μl sample + 3 μlreagent Temperature 5° C. Initial protein concentration: 95 mg/ml Finalprotein concentration: 198 mg/ml Number of days (d), temperature (° C.)well Reagents: 1 d, 5° C. 3 d, 5° C. 8 d, 5° C. 18 d, 5° C. A1 1.8 MNa—K-phosphate pH 5.0 L L C, G A, G A2 1.8 M Na—K-phosphate pH 5.6 L LC, L, A C, L, G A3 1.8 M Na—K-phosphate pH 6.2 L C C C, G A4 1.8 MNa—K-phosphate pH 7.0 L, G L, G L, G G A5 1.8 M Na—K-phosphate pH 7.4 GG G G A6 1.8 M Na—K-phosphate pH 8.2 G G G G B1 2.0 M Na—K-phosphate pH5.0 L L G A, G B2 2.0 M Na—K-phosphate pH 5.6 L, A L, A L, G A, G B3 2.0M Na—K-phosphate pH 6.2 L, C C C, G C, G B4 2.0 M Na—K-phosphate pH 7.0A, G A, G G G B5 2.0 M Na—K-phosphate pH 7.4 A, G A, G G G B6 2.0 MNa—K-phosphate pH 8.2 A, G A, G G G C1 2.2 M Na—K-phosphate pH 5.0 L, AL, A L, A A, G C2 2.2 M Na—K-phosphate pH 5.6 L, A L, A L, A A, G C3 2.2M Na—K-phosphate pH 6.2 L, C C C C, G C4 2.2 M Na—K-phosphate pH 7.0 A,G A, G G G C5 2.2 M Na—K-phosphate pH 7.4 A, G A, G G G C6 2.2 MNa—K-phosphate pH 8.2 A, G A, G G G D1 2.3 M Na—K-phosphate pH 5.0 L, AL, A L, A A, G D2 2.3 M Na—K-phosphate pH 5.6 L L L A, G D3 2.3 MNa—K-phosphate pH 6.2 L, C C C C, G D4 2.3 M Na—K-phosphate pH 7.0 A, GA, G G G D5 2.3 M Na—K-phosphate pH 7.4 A, G A, G G G D6 2.3 MNa—K-phosphate pH 8.2 A, G A, G G GMicroscopy ObservationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsd = daysG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 4 Box code: MCSA35 Sample: GTC hSA: 7F 4AC, concentrated usingdialysis against PEG 20 k. Mix 45 μl hSA concentrate, 45 ml water and 10μl caprylate solution (32 mg/ml pH 7.3.). Final caprylate concentrationis 3.2 mg/ml. Drop: 3 μl sample + 3 μl reagent Temperature 5° C. Initialprotein concentration: 49 mg/ml Final protein concentration: 98 mg/mlNumber of days (d), temperature (° C.) well Reagents: 1 d, +5 C. 2 d, +5C. 3 d, +5 C. 13 d, +5 C. A1 1.8 M Na—K-phosphate pH 5.9 L C, L C, L C,G A2 1.8 M Na—K-phosphate pH 6.0-6.1 L C, L C, L C, G A3 1.8 MNa—K-phosphate pH 6.2 L C, L C, L, G C, G A4 1.8 M Na—K-phosphate pH6.3-6.4 L C, L L, C C, G A5 1.8 M Na—K-phosphate pH 6.4-6.5 L L L C, GA6 1.8 M Na—K-phosphate pH 6.6 L L, A L G B1 2.0 M Na—K-phosphate pH 5.9L C, L C, L C, G B2 2.0 M Na—K-phosphate pH 6.0-6.1 L C, L, A C, L, G C,G B3 2.0 M Na—K-phosphate pH 6.2 L C, L, G C, LG C, G B4 2.0 MNa—K-phosphate pH 6.3-6.4 L L, G L, G G B5 2.0 M Na—K-phosphate pH6.4-6.5 L L, G L, G G B6 2.0 M Na—K-phosphate pH 6.6 L L, G L, G G C12.2 M Na—K-phosphate pH 5.9 L C, L, A C, L, A C, G C2 2.2 MNa—K-phosphate pH 6.0-6.1 L C, L, G C, L, G C, G C3 2.2 M Na—K-phosphatepH 6.2 L, C C, L, G C, L, G C, G C4 2.2 M Na—K-phosphate pH 6.3-6.4 L L,G, C C, L, G, G C5 2.2 M Na—K-phosphate pH 6.4-6.5 L L, G L, G G C6 2.2M Na—K-phosphate pH 6.6 L L, G L, G G D1 2.3 M Na—K-phosphate pH 5.9 LC, L, A C, L, A C, G D2 2.3 M Na—K-phosphate pH 6.0-6.1 L, C C, LG C, L,G C, G D3 2.3 M Na—K-phosphate pH 6.2 L, C C, LG C, L, G C, G D4 2.3 MNa—K-phosphate pH 6.3-6.4 L C, L, G, C, L, G, G D5 2.3 M Na—K-phosphatepH 6.4-6.5 L L, G L, G G D6 2.3 M Na—K-phosphate pH 6.6 L, G L, G L, G GMicroscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsd = daysG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

EXAMPLE 3 Crystallization of Albumin in Impure Starting Material

The following process description is originally made from a recombinanthSA starting material which contains much of impurities which interferethe crystallization. The first process steps are needed for removal ofimpurities. If more pure albumin is used, the number of steps iscorrespondingly reduced.

Step 1. Concentration

The starting material should be concentrated by ultrafiltration filteredas much as possible. High protein concentration will reduce the usage ofphosphate and increase the yield of crystallizable rhSA. The materialshould also be filtered with water in the end of concentration procedurein order to reduce the salts originating from the previous processsteps. Guideline for protein concentration: A280_(nm)=150-200.

Step 2. Initial Precipitation with Phosphate

Add 2.5 volumes of 4 M phosphate (pH 6.2) into 1.5 volumes of rhSAconcentrate. The final phosphate concentration at this step is 2.5 Mwhich precipitates impurities but not albumin. This procedure ispreferably performed at room temperature.

Step 3. Filtration

Remove the precipitate that is filtered out with a Buchner funnel orpressure chamber filter. Diatomaceous earth is used as filter aid, sincethe precipitate is very finely grained amorphous material.Centrifugation is not a convenient option since the precipitate willfloat on the top of the liquid.

Step 4. Crystallization

Crystallization of hSA is started by adding more of 4 M phosphate (pH6.2) in the filtrate until concentration is 2.8 M. Crystallization isperformed at refrigerated temperatures preferably around 5.0° C.Crystallization of the hSA, according to this embodiment of the currentinvention starts spontaneously within 24 hours. In an alternateembodiment the addition of seed crystals will make the process morerapid.

Step 5. Washing of Crystals

According to the preferred embodiment of the current invention crystalsare washed either by centrifugation or preferably by being filtered. Incentrifugation the crystals float on the top of phosphate buffer.Washing is performed with fresh 2.8 M phosphate solution at temperaturearound +5° C. Crystal washing should be made with low pressuredifference, less than 0.1 bar. The washing is repeated 3-4 times untilthe soluble protein of filtrate remains at nearly constant low level,A280_(nm)=1.0 or less.

Step 6. Formulation of Crystal Stock

The washed crystals are dispersed in a small volume of the 2.8 Mphosphate buffer. Crystals can be stored in this until formulated forthe next step.

EXAMPLE 4 Crystallization of Impure Albumin after Heat Treatment

Starting Material

The feedstream material used for this example had had a significantamount of proteins as impurities. Albumin was approximately 30% of theprotein present. As already stated this is within the typical range offeedstream materials supplied from clarified or partially purifiedtransgenic or cell culture sources. The solutions used included 107 mlof 4 M phosphate pH 6.2 was added into 200 ml of starting materialsolution (which was in 2 M phosphate before this step) to get 2.70 Msolution. According to the current invention this precipitated solutionwas used as starting solution for crystallization.

Heat Treatment

The precipitated starting solution (in 2.70 M phosphate) was incubatedat 55° C. for 90 minutes. A substantial amount of rod-like crystals wereformed along with an amorphous precipitate as a result of this heattreatment. Approximately 75% to 80% of total protein was crystallized orprecipitated. The crystals and precipitate were removed by filtering theslurry through glass fiber filter when it was still hot. Somediatomaceous earth was used as a filter aid. Only the supernatantfiltrate containing albumin was taken for the next step.

Concentration and Filtration

After being filtered, the solution had too low albumin concentration forcrystallization. Thus it was concentrated and diafiltrated at 55° C.with a Fresenius Polysulfone UF 6.2 Hemoflow F5HPS dialysis cartridge.The detailed data of process steps is shown in the table 6.

hSA Crystallization

Phosphate concentration of the concentrate solution was increased andadjusted slowly to 2.8. At the same time the batch was gently agitatedin a refrigerator in order to crystallize hSA.

The hSA crystals were harvested and washed by vacuum being filtered.Washing solution was 2.88 M phosphate pH 6.4. Heat crystallization ofimpurities is technically easy and rapid method to remove majorimpurities of the starting material solution as shown in table 5 belowand in FIG. 7. TABLE 5 Purity Analysis of the Starting Material andWashed Crystals. Results of ELISA Assays. Impure Washed Crystal ELISAassay Starting Material Samples 101-98 BSA (ppm) 85000 14300β-lactoglobulin (ppm) 3488000 4500 α-lactalbumin (ppm) 143000 >100 IgG(ppm) 69000 >1700

Starting material: Genzyme Transgenics hSA: albumin concentrate lot # X1131FF, protein concentration assays: TP 84 g/l and 79 g/l, ALB 28.58 g/land 28.75 g/l. TABLE 6 Impure Albumin from Starting Material toCrystals. Protein % of Batch number and Composition of the Volume Totalprot: starting procedure solution, notes (ml) A 280 × volume materialStart with a lot of starting material from 200 14750 100 productConcentrate, Sigma diafiltrate Add 4 M phosphate Phosphate 2.7 M 30714736 43.7 pH 6.2 Heat 90 minutes at strongly precipitated, 307 1473643.7 55° C. mostly crystalline Add filter aid and clear filtrate 2603276 22 filtrate while hot Concentrate with clear concentrate, 111 302420 ultrafiltration 1.9 M phosphate Add 4 M phosphate Phosphate 2.8 M90.8 3024 20 gradually, concentrate Protein in the filtrate 0.45 μmfiltrate of the 90.8 1843 10 crystal slurry Protein in the crystalscrystals calculated by 90.8 1180 8 difference of A280 nm 33.3-20.3Material balance of the whole sequence of steps was calculated using the200 ml aliquot of the sample solution.

EXAMPLE 5 Recrystallization of Albumin by Using Heating And CoolingCycles

Previously made hSA crystal slurry in 2.7 M phosphate pH 6.2 with aprotein concentration of 37.5 g/l was used as starting material for thisstudy conducted according to a preferred embodiment of the currentinvention. Five samples of the crystal slurry, 0.50 ml each, wereincubated alternatively at 40, 45, 55, 65 or 70° C. for 60 minutes.Crystals were dissolved in all the tested temperatures. At the end ofheating, the samples were filtered through 0.45 μm filter. Solubleprotein of the filtrates was determined by measuring absorbance at 280nm.

Approximately 10% of total protein was precipitated when the crystalslurry was incubated at 40-55° C. for an hour as seen in tables 7 and 8and in FIG. 13. This value was rather constant in this temperature rangeand it represents impurities which were removed in the heating beingfiltered procedure. When the temperature was increased from 65 to 70°C., the soluble protein of the sample was decreased from 83% to 37%.Thus, also albumin starts to precipitate irreversibly at 70° C.

After being filtered, samples 1 through 5 were heated up to 65° C. wereall well recrystallized after 18 hours in refrigerator set at 5° C. Whenthe sample 5 was incubated at 70° C., no crystals were produced in thefiltrate, which indicates that albumin is not stable at that temperaturein 2.7 M phosphate, see Table 8. These examples show that albumin can,according to a preferred embodiment of the current invention, be wellpurified by using heating up to 65° C. and thereafter being filtered andgoing through appropriate re-crystallization cycles. TABLE 7 Results ofthe Heat Precipitation Study of hSA Crystal Slurry Soluble A 280 ofprotein Sample Heated Observation after heat treatment, filtrate (%) ofNo: at before being filtered 0.45 μm total 1 40° C. Slightly turbidsolution. Most 17.08 86 of the crystals were dissolved 2 45° C. Slightlyturbid solution, no 17.90 90 crystals. 3 55° C. Slightly turbidsolution, no 17.70 89 crystals. 4 65° C. Increasingly turbid solution,no 16.60 83 crystals. 5 70° C. Strongly precipitated slurry, no 7.42 37crystals

TABLE 8 Observations of the heated and filtered samples, re-crystallizedat low temperature. Filtrate of Microscopy observations aftercrystallization in example No: Heated at refrigerator 1 40° C. Wellformed crystals 2 45° C. Different sizes, large and very small crystals3 55° C. Large crystals and crystal clusters 4 65° C. Crystals andcrystal clusters 5 70° C. Clear solution, no crystals

EXAMPLE 6 Crystallization of Albumin at Higher pH

The studies done according to the current invention show that albumincrystal solubility and crystallization may be dependent on pH. Anotherissue is the separation of caprylate in the presence of phosphatebuffer. The solubility of caprylate increases at higher pH. Thus, evenslightly higher pH would be desirable for optimal crystallizationformation. According to the current invention, trial crystallizationswere made at a range of pH 6.2-6.6 with three different levels ofphosphate molarity. Experimental details are presented in the table 9below.

Evaluation of Results

As seen in Table 9 below, albumin was crystallized very effectively whenpH was increased to the values 6.4 and 6.5. Albumin solubility was verylow above pH 6.4. Unfortunately, at the higher pH levels albumin wascrystallized as very small needles (See FIGS. 10-13). It is likely thatthe crystal size could be developed larger by starting thecrystallization at lower pH 6.2 or 6.3. After achieving nearequilibrium, pH could be adjusted to higher level by gradually adding 4MK₂HPO₄. TABLE 9 Test tube crystallization of albumin concentrate 102-5.Soluble protein 20 h Sample No#. (nd = not 3 ml each exp. determined)phosphate RhSA 31 mg/ml A280 nm M pH 102-9-1 nd 2.46 6.2 N 102-9-2 nd2.46 6.3 N 102-9-3 17.6 2.46 6.4 Initially L, finally C 102-9-4 6.2 2.466.5 L, C, very thin needles 102-9-5 1.5 2.46 6.6 C, very thin needles, A102-9-6 nd 2.57 6.2 Intially L, finally C 102-9-7 16.2 2.57 6.3Initially L, G, finally C 102-9-8 9.0 2.57 6.4 L, G, C needles, 102-9-90.3 2.57 6.5 C, short needles or rods 102-9-10 nd 2.57 6.6 C, shortneedles or rods 102-9-11 nd 2.67 6.2 Initially G, finally C 102-9-1214.9 2.67 6.3 Initially L, G, finally C 102-9-13 0.3 2.67 6.4 C, manyvery small needles 102-9-14 nd 2.67 6.5 C, many very small needles102-9-15 nd 2.67 6.6 C, many very small needlesMicroscopy observationN = no phase separationA = amorphous precipitateC = crystalsL = liquid phase separationG = gel particle separation

EXAMPLE 7 Re-Crystallization and Washing of Albumin Crystals

Re-Crystallization and Washing

The washed crystals (102-12) were heated 5 minutes at 45° C. Thecrystals dissolved rapidly. The solution was filtered with 0.45 μmsyringe filter while still warm. The filtered solution was agitated at2° C. until crystallized (3 days). The batch was crystallized well (FIG.2). The crystals were harvested by being filtered and washed with 2.82 MpH 6.2 phosphate on a 0.45 μm (50 mm diameter) Sartorius membrane. Thewashed crystals were dispersed in 2.82 M phosphate buffer. TABLE 10Recrystallization and Washing of Albumin Crystals Step Ml A 280 nmalbumin g Start with 102-12 26.9 36.9 1.877 Step 1. heat 5 minutes at45° C. 26.9 Step 2. filtrate 0.45 μm 26 Step 3. agitated at 2° C. 3 days26 (see: FIG. 2) Step 4. Harvest and wash, filtrates 37.8 3.6 0.256Crystal suspension in 2.82 M 22.1 34.8 1.451 Sample 102-13 to Sigma 7 1834.8 1.182Indications and Uses

Hypovolemia

Hypovolemia is a possible indication for albumin purified and madeavailable by the method of the current invention, 25% Solution, Buminate25%. Its effectiveness in reversing hypovolemia depends largely upon itsability to draw interstitial fluid into the circulation. It is mosteffective with patients who are well hydrated. When hypovolemia is longstanding and hypoalbuminemia exists accompanied by adequate hydration oredema, 25% albumin is preferable to 5% protein solutions. However, inthe absence of adequate or excessive hydration, 5% protein solutionsshould be used or 25% albumin should be diluted with crystalloid.Although crystalloid solutions and colloid-containing plasma substitutescan be used in emergency treatment of shock, albumin has a prolongedintravascular half-life. When blood volume deficit is the result ofhemorrhage, compatible red blood cells or whole blood should beadministered as quickly as possible.

Hypoalbuminemia

Hypoalbuminemia is another possible indication for use of albuminpurified and made available by the method of the current invention, 25%Solution, Buminate 25%. Hypoalbuminemia can result from one or more ofthe following: Inadequate production (malnutrition, burns, major injury,infections, etc.); Excessive catabolism (burns, major injury,pancreatitis, etc.); Loss from the body (hemorrhage, excessive renalexcretion, burn exudates, etc.); and Redistribution within the body(major surgery, various inflammatory conditions, etc.).

When albumin deficit is the result of excessive protein loss, the effectof administration of albumin will be temporary unless the underlyingdisorder is reversed. In most cases, increased nutritional replacementof amino acids and/or protein with concurrent treatment of theunderlying disorder will restore normal plasma albumin levels moreeffectively than albumin solutions. Occasionally hypoalbuminemiaaccompanying severe injuries, infections or pancreatitis cannot bequickly reversed and nutritional supplements may fail to restore serumalbumin levels. In these cases, albumin (Human), 25% Solution, Buminate25% might be a useful therapeutic adjunct.

Burns

An optimum regimen for the use of albumin, electrolytes and fluid in theearly treatment of burns has not been established, however, inconjunction with appropriate crystalloid therapy, Albumin (Human), 25%Solution, Buminate 25% may be indicated for treatment of oncoticdeficits after the initial 24 hour period following extensive burns andto replace the protein loss which accompanies any severe burn.

Adult Respiratory Distress Syndrome (ARDS)

A characteristic of ARDS is a hypoproteinemic state which may becausally related to the interstitial pulmonary edema. Althoughuncertainty exists concerning the precise indication of albumin infusionin these patients, if there is a pulmonary overload accompanied byhypoalbuminemia, 25% albumin solution may have a therapeutic effect whenused with a diuretic.

Nephrosis

Albumin (Human), 25% Solution may be a useful aid in treating edema inpatients with severe nephrosis who are receiving steroids and/ordiuretics.

Cardiopulmonary Bypass Surgery

Albumin (Human), 25% Solution, Buminate 25% has been recommended priorto or during cardiopulmonary bypass surgery, although no clear dataexist indicating its advantage over crystalloid solutions.

Hemolytic Disease of the Newborn (HDN)

Albumin (Human), 25% Solution, Buminate 25% may be administered in anattempt to bind and detoxify unconjugated bilirubin in infants withsevere HDN.

It is also possible to use the albumin purified by means of the currentinvention as an excipient for the delivery of pharmaceuticals.

Crystal Screening Experiments

The hanging drop screens are made in boxes with 24 wells divided in 4rows (A, B, C, D) and 6 columns. The screens are presented in thepreliminary report only as lists of the reagents as follows. A briefcomment to the results is provided. In the final report the results areprovided in tables with complete details similar to the followingexample tables.

Microscopy and Tables

The sample evaluations by microscopy are made in the beginning ofexperiments with 2-3 days frequency and later on once a week. Themicroscopy observations in the tables are in abbreviated form using thefollowing letters:

-   -   N=no precipitation or other phase separation    -   C=Some crystals present    -   CC=A significant amount of crystals present    -   A=amorphous precipitate, cloud of non-transparent particles        looking like cloud or brownish smoke, size of the particles is        near the lowest limit of separation power of light microscope    -   AA=A significant amount of amorphous precipitate    -   L=liquid phase separation, spherical transparent droplets,        looking like oil in water    -   LL=A significant liquid separation    -   G=gel lumps, irregular glassy transparent particles of several        micrometers in diameter    -   GG=A significant amount of gel

X=contaminated or dried drop, discontinued experiment TABLE 11 Reversetemperature effect with amorphous precipitate. Precipitates increases athigher temperature under microscopy. Crystals were very temperaturesensitive, they dissolve at higher temperature. Start date (d, m, y):17.8.2001 Sample: GTC hSA: labeled 7F-5AC Drop: 8 μl sample + 2 μlreagent Temperatures: +25° C. or +4° C. Initial protein concentration:11.7 mg/ml Final protein concentration: 58 mg/ml Buffer: 0.05 MK—Na-phosphate pH 7.4 well Reagents 3 d +25° C. 3 d +4° C. 8 d, +4° C.27 d, +4° C. A1 17% PEG 3350 0.08% decanol N N L, G N A2 18% PEG 33500.08% decanol N N L N A3 19% PEG 3350 0.08% decanol N N C CC A4 22% PEG3350 0.08% decanol N N L, G N A5 25% PEG 3350 0.08% decanol N N G N A630% PEG 3350 0.08% decanol AA A, GG G GG B1 17% PEG 3350 0.36% decanol NN N N B2 18% PEG 3350 0.36% decanol N N N N B3 19% PEG 3350 0.36%decanol N N N N B4 22% PEG 3350 0.36% decanol N N A N B5 25% PEG 33500.36% decanol N L, GG G N B6 30% PEG 3350 0.36% decanol AA A L, G GG, LLC1 1.5 M (NH₄)₂SO₄ 0.08% decanol N N N N C2 1.65M (NH₄)₂SO₄ 0.08%decanol N N N N C3 1.8 M (NH₄)₂SO₄ 0.08% decanol N N N N C4 2.1 M(NH₄)₂SO₄ 0.08% decanol N N N N C5 2.4 M (NH₄)₂SO₄ 0.08% decanol G A, GGC, A C, L C6 2.7 M (NH₄)₂SO₄ 0.08% decanol GG CC, G CC, G CC, G D1 1.5 M(NH₄)₂SO₄ 0.36% decanol N N N N D2 1.65M (NH₄)₂SO₄ 0.36% decanol N N N ND3 1.8 M (NH₄)₂SO₄ 0.36% decanol N N N N D4 2.1 M (NH₄)₂SO₄ 0.36%decanol N A N N D5 2.4 M (NH₄)₂SO₄ 0.36% decanol AA C, G L LL D6 2.7 M(NH₄)₂SO₄ 0.36% decanol L, A, GG CC, G C, G CC, GMCS 23 AAT, Sample: GTC hSA: labeled 7F-5AC; PEG 3350 or (NH₄)₂SO₄buffered with 0.05 M K—Na-phosphate pH 7.4 effect of decanol additive.Row A: 500 μl of PEG 3350 17, 18, 19, 22, 25 or 30% in 0.05 MK—Na-phosphate pH 7.4; add 10 μl 4% decanol to each 500 μl of A reagentsto get final 0.08% decanol. Row B: like row A, but add 50 μl 4% decanolto each# 500 μl of A reagents to get final 0.36% decanol. Row C: 500 μl of(NH₄)₂SO₄ 1.5, 1.65, 1.8, 2.1, 2.4 or 2.7 M in 0.05 M K—Na-phosphate pH7.4; add 10 μl 4% decanol to each 500 μl of A reagents to get final0.08% decanol. Row D: like row C, but add 50 μl 4% decanol to each 500μl of A reagents to get final 0.36% decanol.Results: Crystals of high quality (FIG. 14) were produced with 2.4M and2.7M ammonium sulfate-decanol combinations Crystals of a different habitwere produced with 19% PEG, 0.08% decanol. Amorphous or gel precipitateswere also produced with both reagents. Crystallization is verycritically related to the reagent concentration and temperature.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 12 Start date (d, m, y): 17.8.2001 Sample: Sigma A-9511 Drop: 2 μlsample + 2 μl reagent Temperatures: +25° C. or +7° C. Initial proteinconcentration: 55 mg/ml Final protein concentration: 110 mg/ml Buffer:0.05 M K—Na-phosphate pH 7 well Reagents 3 d +25° C. 3 d +7° C. 8 d, +7°C. 27 d, +7° C. A1 17% PEG 3350 0.08% decanol N N G N A2 18% PEG 33500.08% decanol N N L A A3 19% PEG 3350 0.08% decanol N N A N A4 22% PEG3350 0.08% decanol N N A N A5 25% PEG 3350 0.08% decanol N N A N A6 30%PEG 3350 0.08% decanol AA AA A AA B1 17% PEG 3350 0.36% decanol N N N NB2 18% PEG 3350 0.36% decanol N N N N B3 19% PEG 3350 0.36% decanol N NN N B4 22% PEG 3350 0.36% decanol N N A N B5 25% PEG 3350 0.36% decanolN N N N B6 30% PEG 3350 0.36% decanol N N L, G GG C1 1.5 M (NH₄)₂SO₄0.08% decanol N N N N C2 1.65M (NH₄)₂SO₄ 0.08% decanol N N N N C3 1.8 M(NH₄)₂SO₄ 0.08% decanol N N N N C4 2.1 M (NH₄)₂SO₄ 0.08% decanol N A A NC5 2.4 M (NH₄)₂SO₄ 0.08% decanol GG GG GG LL, AA C6 2.7 M (NH₄)₂SO₄0.08% decanol GG GG GG GG D1 1.5 M (NH₄)₂SO₄ 0.36% decanol GG GG C C D21.65M (NH₄)₂SO₄ 0.36% decanol N N N N D3 1.8 M (NH₄)₂SO₄ 0.36% decanol NN N N D4 2.1 M (NH₄)₂SO₄ 0.36% decanol N A N N D5 2.4 M (NH₄)₂SO₄ 0.36%decanol N A L L, A, G D6 2.7 M (NH₄)₂SO₄ 0.36% decanol LL, G LL, GG GGGGTable 12 above, MCS 22 AAS, Sample: Sigma hSA, A-9511PEG 3350 or (NH₄)₂SO₄ buffered with 0.05 M K—Na-phosphate pH 7.4effect of decanol additive Similar screen to table 11.Results: Sigma hSA did not crystallize as well as GTC hSA 7F-5AC. Onlyone sample in 1.5 M (NH₄)₂SO₄, 0.36% decanol produced high qualitycrystals.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 13 Reverse temperature effect with amorphous precipitate.precipitates increase at higher temperature under microscopy. Crystalswere very temperature sensitive, they dissolve at higher temperature.Box code: MCS 22AT Start date (d, m, y): 17.8.2001 Sample: GTC hSA:labeled 7F-5AC Drop: 8 μl sample + 2 μl d reagent Temperatures: +25° C.or +7° C. Initial protein concentration: 11.7 mg/ml Final proteinconcentration: 58 mg/ml Buffer: 0.05 M K—Na-phosphate pH 7.4 1 d 30 d,well Reagents +25° C. 4 d +25° C. +7° C. d, ° C. A1 17% PEG 3350 N N NA2 18% PEG 3350 N N N A3 19% PEG 3350 N N N A4 22% PEG 3350 N N A A5 25%PEG 3350 N N N A6 30% PEG 3350 LL LL A, G B1 17% PEG 3350 0.4% caprylicacid N N A B2 18% PEG 3350 0.4% caprylic acid N N A B3 19% PEG 3350 0.4%caprylic acid N N A B4 22% PEG 3350 0.4% caprylic acid N N N B5 25% PEG3350 0.4% caprylic acid N N A, L B6 30% PEG 3350 0.4% caprylic acid LLLL A, L C1 1.5 M (NH₄)₂SO₄ N N N C2 1.65M (NH₄)₂SO₄ N N N C3 1.8 M(NH₄)₂SO₄ N N N C4 2.1 M (NH₄)₂SO₄ N N A C5 2.4 M (NH₄)₂SO₄ A N A, G C62.7 M (NH₄)₂SO₄ GG C, G A, G D1 1.5 M (NH₄)₂SO₄ 0.4% caprylic acid N N ND2 1.65M (NH₄)₂SO₄ 0.4% caprylic acid N N N D3 1.8 M (NH₄)₂SO₄ 0.4%caprylic acid N N N D4 2.1 M (NH₄)₂SO₄ caprylic acid N N A D5 2.4 M(NH₄)₂SO₄ caprylic acid N LL, GG A D6 2.7 M (NH₄)₂SO₄ 0.4% caprylic acidL, G LL, GG AFor table 13 MCS 22 AT, Sample: GTC hSA: labeled 7F-5AC PEG 3350 or(NH₄)₂SO₄ buffered with 0.05 M K—Na-phosphate pH 7.4 effect of caprylicacid additive.Results: Unstable crystals were produced in only 2.7 M (NH₄)₂SO₄ withoutcaprylic acid.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 14 Start date (d, m, y): 17.8.2001 Sample: Sigma A-9511 Drop: 2 μlsample + 2 μl reagent Temperatures: +25° C. or +7° C. Initial proteinconcentration: 55 mg/ml Final protein concentration: 110 mg/ml Buffer:0.05 M K—Na-phosphate pH 7.4 well Reagents 1 d +25° C. 4 d +25° C. 30 d,+7° C. d, ° C. A1 17% PEG 3350 N N A, G A2 18% PEG 3350 N N N A3 19% PEG3350 N X X A4 22% PEG 3350 N X X A5 25% PEG 3350 N X X A6 30% PEG 3350GG AA X B1 17% PEG 3350 0.4% caprylic acid N N A, G B2 18% PEG 3350 0.4%caprylic acid N N A, G B3 19% PEG 3350 0.4% caprylic acid N N A, G B422% PEG 3350 0.4% caprylic acid N N N B5 25% PEG 3350 0.4% caprylic acidN N N B6 30% PEG 3350 0.4% caprylic acid A A N C1 1.5 M (NH₄)₂SO₄ N N NC2 1.65M (NH₄)₂SO₄ N N N C3 1.8 M (NH₄)₂SO₄ N N N C4 2.1 M (NH₄)₂SO₄ A NA C5 2.4 M (NH₄)₂SO₄ GG GG AA C6 2.7 M (NH₄)₂SO₄ GG GG AA D1 1.5 M(NH₄)₂SO₄ 0.4% caprylic acid N N N D2 1.65M (NH₄)₂SO₄ 0.4% caprylic acidN N N D3 1.8 M (NH₄)₂SO₄ 0.4% caprylic acid N N N D4 2.1 M (NH₄)₂SO₄0.4% caprylic acid A, G A, G A, G D5 2.4 M (NH₄)₂SO₄ 0.4% caprylic acidA, G GG AA D6 2.7 M (NH₄)₂SO₄ 0.4% caprylic acid GG GG AAMCS 22 AT, Sample: Sigma hSA, A-9511 PEG 3350 or (NH₄)₂SO₄ buffered with0.05 M K—Na-phosphate pH 7.4 effect of caprylic acid additive. Similarscreen to the table 3.Results: No crystals produced. Only precipites at the higher reagentconcentrations.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 15 Start date (d, m, y): 10.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent Temperatures +7° C. Initial proteinconcentration: 12.2 mg/ml Final protein concentration: 73 mg/ml Buffers:0.1 M Na—K-phosphates pH 5.0-7.0 number of days (d), temperature (° C.)well Reagents 3 d, +7° C. 8 d, +7° C. 10 d, +7° C. 40 d, +7° C. A1 1.0 M(NH₄)₂SO₄ pH 5.0 N A A, G A, G A2 1.0 M (NH₄)₂SO₄ pH 5.6 N N N N A3 1.0M (NH₄)₂SO₄ pH 5.9 N N N N A4 1.0 M (NH₄)₂SO₄ pH 6.2 N N N N A5 1.0 M(NH₄)₂SO₄ pH 6.6 N N N N A6 1.0 M (NH₄)₂SO₄ pH 7.0 N N N N B1 1.5 M(NH₄)₂SO₄ pH 5.0 N AA, GG G A, G B2 1.5 M (NH₄)₂SO₄ pH 5.6 N N A A B31.5 M (NH₄)₂SO₄ pH 5.9 N N A A B4 1.5 M (NH₄)₂SO₄ pH 6.2 N N A A B5 1.5M (NH₄)₂SO₄ pH 6.6 N N A A B6 1.5 M (NH₄)₂SO₄ pH 7.0 N N A A C1 2.0 M(NH₄)₂SO₄ pH 5.0 AA AA GG A, G C2 2.0 M (NH₄)₂SO₄ pH 5.6 N AA, G GG A C32.0 M (NH₄)₂SO₄ pH 5.9 N AA, C GG A C4 2.0 M (NH₄)₂SO₄ pH 6.2 AA, C AAGG, C A C5 2.0 M (NH₄)₂SO₄ pH 6.6 A A CC A C6 2.0 M (NH₄)₂SO₄ pH 7.0 A ACC A D1 3.0 M (NH₄)₂SO₄ pH 5.0 AA, L AA, GG GG A, G D2 3.0 M (NH₄)₂SO₄pH 5.6 AA, L AA, GG GG A D3 3.0 M (NH₄)₂SO₄ pH 5.9 AA, L AA, GG GG A, GD4 3.0 M (NH₄)₂SO₄ pH 6.2 A AA, GG GG A D5 3.0 M (NH₄)₂SO₄ pH 6.6 A AA,GG GG A, G D6 3.0 M (NH₄)₂SO₄ pH 7.0 AA, L AA, GG GG A, GMCS16 Sample: GTC hSA: labeled 7F-5AC Ammonium sulfate buffered withphosphates 4 rows: A 1.0 M, B 1.5 M, C 2.0 M, D 3.0 M 6 columns: 0.1 Mphosphates pH 5.0 pH 5.6 pH 5.9 pH 6.2 pH 6.6 pH 7.0Results: Amorphous precipitates above 2M. Some unstable poor qualitycrystals which disappeared in 40 d storage.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 16 Start date (d, m, y): 10.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent Temperatures +7° C. Initial proteinconcentration: 12.2 mg/ml Final protein concentration: 73 mg/ml Buffers:0.1 M Na—K-phosphates pH 5.0-7.0 number of days (d), temperature (° C.)well Reagents 3 d, +7° C. 6 d, +7° C. 10 d, +7° C. 40 d, +7° C. A1 1.0 Msodium sulfate pH 5.0 N N N N A2 1.0 M sodium sulfate pH 5.6 N N N N A31.0 M sodium sulfate pH 5.9 N N N N A4 1.0 M sodium sulfate pH 6.2 N N NN A5 1.0 M sodium sulfate pH 6.6 N N N N A6 1.0 M sodium sulfate pH 7.0N N N N B1 1.5 M sodium sulfate pH 5.0 N N AA A B2 1.5 M sodium sulfatepH 5.6 N N AA A B3 1.5 M sodium sulfate pH 5.9 N N AA A B4 1.5 M sodiumsulfate pH 6.2 N N AA A B5 1.5 M sodium sulfate pH 6.6 N N AA A B6 1.5 Msodium sulfate pH 7.0 N N AA A C1 1.75 M sodium sulfate pH 5.0 N G, C AAA C2 1.75 M sodium sulfate pH 5.6 N A, G AA A C3 1.75 M sodium sulfatepH 5.9 N A AA A C4 1.75 M sodium sulfate pH 6.2 N N AA A C5 1.75 Msodium sulfate pH 6.6 N N AA A C6 1.75 M sodium sulfate pH 7.0 N N AA AD1 2.0 M sodium sulfate pH 5.0 AA, L GG, C LL, GG A D2 2.0 M sodiumsulfate pH 5.6 AA, L GG LL, GG A D3 2.0 M sodium sulfate pH 5.9 AA, LGG, C A, GG A D4 2.0 M sodium sulfate pH 6.2 AA, LL GG, L, C L, GG G, AD5 2.0 M sodium sulfate pH 6.6 AA, LL G, L, C L, GG G, A D6 2.0 M sodiumsulfate pH 7.0 A, LL GG AA G, AMCS17, Sample: GTC hSA: labeled 7F-5AC Sodium sulfate buffered withphosphates 4 rows: A 1.0 M, B 1.5 M, C 1.75 M, D 2.0 M Na₂SO₄ 6 columns:0.1 M phosphates pH 5.0 pH 5.6 pH 5.9 pH 6.2 pH 6.6 pH 7.0Results: Unstable crystals in 1.75 M and 2.0 M Na₂SO₄ Precipitation,amorphous and gel in 1.5-2.0 M Na₂SO₄Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.Note:Crystals were not stable.

TABLE 17 Start date (d, m, y): 28.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 2 μl reagent Temperatures +7° C. Initial proteinconcentration: 10.3 mg/ml Final protein concentration: 36 mg/ml Buffers:0.1 M Na—K-phosphates pH 6.6-7.8 number of days (d), temperature (° C.)Reagents: 12 d, 22 d, well 0.36% decanol in all samples +7° C. +7° C. 28d, +7° C. A1 1.6 M Na—K-phosphate pH 6.6 L N N A2 1.6 M Na—K-phosphatepH 7.0 L N N A3 1.6 M Na—K-phosphate pH 7.2 L N N A4 1.6 MNa—K-phosphate pH 7.4 L N N A5 1.6 M Na—K-phosphate pH 7.6 L N N A6 1.6M Na—K-phosphate pH 7.8 G N N B1 1.8 M Na—K-phosphate pH 6.6 L N N B21.8 M Na—K-phosphate pH 7.0 L N N B3 1.8 M Na—K-phosphate pH 7.2 N N NB4 1.8 M Na—K-phosphate pH 7.4 N N N B5 1.8 M Na—K-phosphate pH 7.6 N NN B6 1.8 M Na—K-phosphate pH 7.8 L, G N N C1 2.0 M Na—K-phosphate pH 6.6L N N C2 2.0 M Na—K-phosphate pH 7.0 L N N C3 2.0 M Na—K-phosphate pH7.2 L N N C4 2.0 M Na—K-phosphate pH 7.4 L N N C5 2.0 M Na—K-phosphatepH 7.6 L N N C6 2.0 M Na—K-phosphate pH 7.8 L, G N N D1 2.2 MNa—K-phosphate pH 6.6 L N L D2 2.2 M Na—K-phosphate pH 7.0 CC N L D3 2.2M Na—K-phosphate pH 7.2 CC N L D4 2.2 M Na—K-phosphate pH 7.4 CC N L D52.2 M Na—K-phosphate pH 7.6 CC N CC, L D6 2.2 M Na—K-phosphate pH 7.8 CCN CC, LMCSA30, Sample: GTC hSA: labeled 7F-5AC1.6-2.2 M K—Na-phosphates pH 6.6-7.8, 0.36% decanol in all dropsResults: Good quality crystals in 2.2 M phosphates pH 7.0-7.8. Thecrystals were very temperature sensitive, they dissolved aftermicroscopy at room temperature and crystallized again in refrigerator.Note:Crystals were not stable when moved to room temperature for microscopy.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 18 Start date (d, m, y): 10.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent temperatures +7° C. Initial proteinconcentration: 12.2 mg/ml Final protein concentration: 73 mg/ml Buffers:Na—K-phosphates pH 4.3-6.2 pH number of days (d), temperature (° C.)well Reagents 3 d, +7° C. 8 d, +7° C. 10 d, +7° C. 40 d, +7° C. A1 1.2 MNa—K-phosphate pH 4.3 N N N G, A A2 1.2 M Na—K-phosphate pH 5.0 N N N NA3 1.2 M Na—K-phosphate pH 5.3 N N N N A4 1.2 M Na—K-phosphate pH 5.6 NN N N A5 1.2 M Na—K-phosphate pH 5.9 N N N N A6 1.2 M Na—K-phosphate pH6.2 N N N N B1 1.6 M Na—K-phosphate pH 4.3 N A A G, A B2 1.6 MNa—K-phosphate pH 5.0 N N A G, A B3 1.6 M Na—K-phosphate pH 5.3 N N A AB4 1.6 M Na—K-phosphate pH 5.6 N N N N B5 1.6 M Na—K-phosphate pH 5.9 NN N A B6 1.6 M Na—K-phosphate pH 6.2 N N N A C1 1.8 M Na—K-phosphate pH4.3 N AA, GG A A, G C2 1.8 M Na—K-phosphate pH 5.0 N AA A A, G C3 1.8 MNa—K-phosphate pH 5.3 N N A A C4 1.8 M Na—K-phosphate pH 5.6 N N A A C51.8 M Na—K-phosphate pH 5.9 N N A A C6 1.8 M Na—K-phosphate pH 6.2 N N AA D1 2.2 M Na—K-phosphate pH 4.3 AA AA, GG GG A, G D2 2.2 MNa—K-phosphate pH 5.0 N AA AA A, G D3 2.2 M Na—K-phosphate pH 5.3 N GG,C G A, G D4 2.2 M Na—K-phosphate pH 5.6 N N A A D5 2.2 M Na—K-phosphatepH 5.9 N N A A D6 2.2 M Na—K-phosphate pH 6.2 N N A, G A, GNotes:Reverse temperature effect; amorphous precipitate increases at highertemperature under microscopy. Crystals were not stable, they dissolvedat higher temperature under microscopy.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 19 Start date (d, m, y): 10.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent Temperatures +7° C. Initial proteinconcentration: 12.2 mg/ml Final protein concentration: 73 mg/ml Buffers:Na—K-phosphates pH 6.6-8.2 number of days (d), temperature (° C.) wellReagents: 3 d, +7° C. 8 d, +7° C. 10 d, +7° C. 40 d, +7° C. A1 1.2 MNa—K-phosphate pH 6.6 N N N N A2 1.2 M Na—K-phosphate pH 7.0 N N N N A31.2 M Na—K-phosphate pH 7.4 N N N N A4 1.2 M Na—K-phosphate pH 7.7 N N NN A5 1.2 M Na—K-phosphate pH 8.2 N N N N A6 B1 1.6 M Na—K-phosphate pH6.6 N N A A B2 1.6 M Na—K-phosphate pH 7.0 N N A A B3 1.6 MNa—K-phosphate pH 7.4 A AA, GG A A B4 1.6 M Na—K-phosphate pH 7.7 N N NN B5 1.6 M Na—K-phosphate pH 8.2 N N N N B6 C1 1.8 M Na—K-phosphate pH6.6 N N A A C2 1.8 M Na—K-phosphate pH 7.0 N N A A C3 1.8 MNa—K-phosphate pH 7.4 AA AA, GG CC, GG AA C4 1.8 M Na—K-phosphate pH 7.7N AA GG A C5 1.8 M Na—K-phosphate pH 8.2 N AA GG A C6 D1 2.2 MNa—K-phosphate pH 6.6 A CC, A GG G, A D2 2.2 M Na—K-phosphate pH 7.0 ACC, G GG G, A D3 2.2 M Na—K-phosphate pH 7.4 AA, L GG, LL GG G, A D4 2.2M Na—K-phosphate pH 7.7 AA AA GG AA D5 2.2 M Na—K-phosphate pH 8.2 AA AAGG AA D6Tables 18 and 19 MCS 15/1 and MCS 15/2, Sample: GTC hSA: labeled 7F-5ACPhosphates without any other additivespH 4.3-8.2 Na—K-PO₄ concentration 1.2 M-2.2 M4 rows: A 1.2 M, B 1.6 M, C 1.8 M, D 2.2 M11 columns: pH 4.3(NaH₂PO₄) containing: pH 5.0 pH 5.3 pH 5.6 pH 5.9 pH6.2 pH 6.6 pH 7.0 pH 7.4 pH 7.7 pH 8.2.Results: Sharp optimum for precipitate at pH 7.4. Unstable crystalsabove pH 6.6 and at pH 5.3. Much potential for further study.Note:reverse temperature effect; amorphous precipitates increase at highertemperture under microscopy. Crystals were not stable, they dissolved athigher temperature under microscopy.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 20 Start date (d, m, y): 14.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent Temperatures +25° C. or 7° C. Initialprotein concentration: 12.2 mg/ml Final protein concentration: 73 mg/mlBuffers: 0.03 M Na-HEPES pH 7.5-7.8 number of days (d), temperature (°C.) 6 d, well Reagents +25° C. 30 d, +7° C. A1   25% 2-propanol, 20 mMMgCl₂ N N A2   30% 2-propanol, 20 mM MgCl₂ G G, A A3   35% 2-propanol,20 mM MgCl₂ G L, A A4  1.5% benzyl alcohol, 20 mM MgCl₂ N A A5  1.5%benzyl alcohol, 50 mM MgCl₂ N N A6  1.5% benzyl alcohol, 100 mM MgCl₂ NN B1   25% 2-propanol, 100 mM MgCl₂ N N B2   30% 2-propanol, 100 mMMgCl₂ G G, A B3   35% 2-propanol, 100 mM MgCl₂ L L, A B4  1.5% benzylalcohol, 200 mM MgCl₂ N N B5  1.5% benzyl alcohol, 300 mM MgCl₂ N N B6 1.5% benzyl alcohol, 400 mM MgCl₂ N N C1   25% 2-propanol, 200 mM MgCl₂N N C2   30% 2-propanol, 200 mM MgCl₂ G G, A C3   35% 2-propanol, 200 mMMgCl₂ G G, A C4 1.25% benzyl alcohol, 20 mM MgCl₂ N N C5 1.25% benzylalcohol, 100 mM MgCl₂ N N C6 1.25% benzyl alcohol, 300 mM MgCl₂ N N D1  25% 2-propanol, 300 mM MgCl₂ N N D2   30% 2-propanol, 300 mM MgCl₂ N ND3   35% 2-propanol, 300 mM MgCl₂ N N D4  1.0% benzyl alcohol, 20 mMMgCl₂ N N D5  1.0% benzyl alcohol, 100 mM MgCl₂ N N D6  1.0% benzylalcohol, 300 mM MgCl₂ N NMCS21, Sample: GTC hSA: labeled 7F-5ACReagent combinations with magnesium chloride and containing 2-propanolor benzyl alcoholResults: Only precipitates with 2-propanol conatining samples. Noeffects with benzyl alcohol.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 21 Start date (d, m, y): 10.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent temperatures +7° C. Initial proteinconcentration: 12.2 mg/ml Final protein concentration: 73 mg/ml Buffers:0.1 M Na—K-phosphates pH 5.0-7.0 number of days (d), temperature (° C.)3 d, 4 d, 10 d, well Reagents +7° C. +7° C. +7° C. 40 d, +7° C. A1 1.5 MNaCl pH 5.0 N N N N A2 1.5 M NaCl pH 5.6 N N N N A3 1.5 M NaCl pH 5.9 NN N N A4 1.5 M NaCl pH 6.2 N N N N A5 1.5 M NaCl pH 6.6 N N N N A6 1.5 MNaCl pH 7.0 N N N N B1 2.0 M NaCl pH 5.0 N N N N B2 2.0 M NaCl pH 5.6 NN N N B3 2.0 M NaCl pH 5.9 N N N N B4 2.0 M NaCl pH 6.2 N N N N B5 2.0 MNaCl pH 6.6 N N N N B6 2.0 M NaCl pH 7.0 N N N N C1 3.0 M NaCl pH 5.0 NN N N C2 3.0 M NaCl pH 5.6 N N N N C3 3.0 M NaCl pH 5.9 N N N N C4 3.0 MNaCl pH 6.2 N N N N C5 3.0 M NaCl pH 6.6 N N N N C6 3.0 M NaCl pH 7.0 NN N N D1 4.0 M NaCl pH 5.0 N N N N D2 4.0 M NaCl pH 5.6 N N N N D3 4.0 MNaCl pH 5.9 N N N N D4 4.0 M NaCl pH 6.2 N N N N D5 4.0 M NaCl pH 6.6 NN N N D6 4.0 M NaCl pH 7.0 N N N NSample: GTC hSA: labeled 7F-5AC Sodium chloride buffered with phosphates4 rows: A 1.5 M, B 2.0 M, C 3.0 M, D 4.0 M6 columns: 0.1 M phosphates pH 5.0 pH 5.6 pH 5.9 pH 6.2 pH 6.6 pH 7.0Results: No precipitates or phase separationsMicroscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 22 Start date (d, m, y): 10.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent Temperature: +7° C. Initial proteinconcentration: 12.2 mg/ml Final protein concentration: 73 mg/ml Buffers:0.1 M Na—K-phosphates pH 5.0-7.0 number of days (d), temperature (° C.)3 d, 4 d, 10 d, well Reagents +7° C. +7° C. +7° C. 40 d, +7° C. A1 1.5 MKCl pH 5.0 N N N N A2 1.5 M KCl pH 5.6 N N N N A3 1.5 M KCl pH 5.9 N N NN A4 1.5 M KCl pH 6.2 N N N N A5 1.5 M KCl pH 6.6 N N N N A6 1.5 M KClpH 7.0 N N N N B1 2.0 M KCl pH 5.0 N N N N B2 2.0 M KCl pH 5.6 N N N NB3 2.0 M KCl pH 5.9 N N N N B4 2.0 M KCl pH 6.2 N N N N B5 2.0 M KCl pH6.6 N N N N B6 2.0 M KCl pH 7.0 N N N N C1 3.0 M KCl pH 5.0 N N N N C23.0 M KCl pH 5.6 N N N N C3 3.0 M KCl pH 5.9 N N N N C4 3.0 M KCl pH 6.2N N N N C5 3.0 M KCl pH 6.6 N N N N C6 3.0 M KCl pH 7.0 N N N N D1 3.9 MKCl pH 5.0 N N N N D2 3.9 M KCl pH 5.6 N N N N D3 3.9 M KCl pH 5.9 N N NN D4 3.9 M KCl pH 6.2 N N N G, A D5 3.9 M KCl pH 6.6 N N N G, A D6 3.9 MKCl pH 7.0 N N N NSample: GTC hSA: labeled 7F-5AC Potassium chloride buffered withphosphates4 rows: A 1.5 M, B 2.0 M, C 3.0 M, D 4.0 M6 columns: 0.1 M phosphates pH 5.0 pH 5.6 pH 5.9 pH 6.2 pH 6.6 pH 7.0Results: Mostly no precipitates or phase separations. Gel precipitatesafter 40 d in 3.9 M KCl pH 6.2-6.6.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 23 Start date (d, m, y): 15.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent temperatures +25° C. or +4° C. Initialprotein concentration: 12.2 mg/ml Final protein concentration: 73 mg/mlBuffers: 0.035 M TrisHCl pH 8.0 or 8.4 date or number of days(temperature) 5 d, 30 d, well Reagents +25° C. +4° C. A1 10% PEG 6000 50mM NH₄-acetate pH 8.0 N N A2 10% PEG 6000 100 mM NH₄-acetate pH 8.0 N NA3 10% PEG 6000 300 mM NH₄-acetate pH 8.0 N N A4 10% PEG 6000 50 mMNH₄-acetate pH 8.4 A, G N A5 10% PEG 6000 100 mM NH₄-acetate pH 8.4 A, GA A6 10% PEG 6000 300 mM NH₄-acetate pH 8.4 N N B1 15% PEG 6000 50 mMNH₄-acetate pH 8.0 N N B2 15% PEG 6000 100 mM NH₄-acetate pH 8.0 A N B315% PEG 6000 300 mM NH₄-acetate pH 8.0 LL L, A B4 15% PEG 6000 50 mMNH₄-acetate pH 8.4 A, G N B5 15% PEG 6000 100 mM NH₄-acetate pH 8.4 A, GA B6 15% PEG 6000 300 mM NH₄-acetate pH 8.4 N N C1 20% PEG 6000 50 mMNH₄-acetate pH 8.0 A, L X C2 20% PEG 6000 100 mM NH₄-acetate pH 8.0 N AC3 20% PEG 6000 300 mM NH₄-acetate pH 8.0 L L, A C4 20% PEG 6000 50 mMNH₄-acetate pH 8.4 L A, G C5 20% PEG 6000 100 mM NH₄-acetate pH 8.4 G A,G C6 20% PEG 6000 300 mM NH₄-acetate pH 8.4 L L D1 25% PEG 6000 50 mMNH₄-acetate pH 8.0 LL L, G D2 25% PEG 6000 100 mM NH₄-acetate pH 8.0 LLL, G D3 25% PEG 6000 300 mM NH₄-acetate pH 8.0 LL L, G D4 25% PEG 600050 mM NH₄-acetate pH 8.4 LL A, G D5 25% PEG 6000 100 mM NH₄-acetate pH8.4 LL L, G, A D6 25% PEG 6000 300 mM NH₄-acetate pH 8.4 L L, GSample: GTC hSA: labeled 7F-5AC 10-25% PEG 6000, 0.05-0.3 M ammoniumacetate, 0.035 M Tris-HCl pH 8.0-8.4,Results: No crystals. Various precipitates in most of the samples.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 24 Start date (d, m, y): 14.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent temperatures: +25° C. or +4° C. Initialprotein concentration: 12.2 mg/ml Final protein concentration: 73 mg/mlBuffer: 0.1M phosphate pH 74 4 d, well Reagents +25° C. 10 d, +4° C. 30d, +4° C. d, ° C. A1 10% PEG 400 N N A A2 10% PEG 600 N N N A3 10% PEG1000 N N N A4 10% PEG 4000 N N N A5 10% PEG 6000 N N A A6 10% PEG 20,000N N N B1 20% PEG 400 G A A B2 20% PEG 600 N N N B3 20% PEG 1000 N N N B420% PEG 4000 N N N B5 20% PEG 6000 N N N B6 20% PEG 20 000 N N A C1 30%PEG 400 N A N C2 30% PEG 600 N N L C3 30% PEG 1000 N A N C4 30% PEG 4000A, G, L N N C5 30% PEG 6000 N N N C6 30% PEG 20 000 N N A D1 40% PEG 400N N N D2 40% PEG 600 L L L D3 40% PEG 1000 N N N D4 40% PEG 4000 N N ND5 40% PEG 6000 L N N D6 40% PEG 20 000 LL A, G A, GSample: GTC hSA: labeled 7F-5AC PEG of various molecular weights 400-20000, phosphate buffer pH 7.4 in the sample.Results: No crystals and mostly no precipitates, amorphous forms at thehighest concentration of PEG 6000.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.

TABLE 25 Start date (d, m, y): 15.8.2001 Sample: GTC hSA: labeled 7F-5ACDrop: 5 μl sample + 1 μl reagent Temperatures +25° C. or +4° C. Initialprotein concentration: 12.2 mg/ml Final protein concentration: 73 mg/mlBuffers: 0.1 M K—Na-phosphates pH 4.8-8.2 well Reagents 5 d, +25° C. 30d, +4° C. d, ° C. d, ° C. A1 25% PEG 6000 10% 2-propanol pH4.8 LL L, G,A A2 25% PEG 6000 10% 2-propanol pH5.3 LL L, G, A A3 25% PEG 6000 10%2-propanol pH6.2 LL L, G, A A4 25% PEG 6000 10% 2-propanol pH7.0 LL L,G, A A5 25% PEG 6000 10% 2-propanol pH7.4 LL L, G, A A6 25% PEG 6000 10%2-propanol pH8.2 L L, A B1 25% PEG 6000 15% 2-propanol pH4.8 LL L, G, AB2 25% PEG 6000 15% 2-propanol pH5.3 LL L, G, A B3 25% PEG 6000 15%2-propanol pH6.2 LL L, G, A B4 25% PEG 6000 10% 2-propanol pH7.0 LL L,G, A B5 25% PEG 6000 15% 2-propanol pH7.4 L L, G, A B6 25% PEG 6000 15%2-propanol pH8.2 L L, G, A C1 25% PEG 6000 20% 2-propanol pH4.8 LL L, G,A C2 25% PEG 6000 20% 2-propanol pH5.3 LL L, G, A C3 25% PEG 6000 20%2-propanol pH6.2 LL L, G, A C4 25% PEG 6000 20% 2-propanol pH7.0 LL L, AC5 25% PEG 6000 20% 2-propanol pH7.4 LL L, A C6 25% PEG 6000 20%2-propanol pH8.2 L L, A D1 25% PEG 6000 30% 2-propanol pH4.8 L, G X D225% PEG 6000 30% 2-propanol pH5.3 L L, G D3 25% PEG 6000 30% 2-propanolpH6.2 LG L, G, A D4 25% PEG 6000 30% 2-propanol pH7.0 L L, A D5 25% PEG6000 30% 2-propanol pH7.4 LG A, G D6 25% PEG 6000 30% 2-propanol pH8.2LG XSample: GTC hSA: labeled 7F-5AC 25% PEG 6000 and 10-30% 2-propanolbuffered with 0.1 M K—Na-phosphates pH 4.8-8.2row A: 25% PEG 6000 and 10% 2-propanol with buffers pH 4.8, 5.3, 6.2,7.0, 7.4 and 8.2row B: 25% PEG 6000 and 15% 2-propanol with buffers pH 4.8, 5.3, 6.2,7.0, 7.4 and 8.2row C: 25% PEG 6000 and 20% 2-propanol with buffers pH 4.8, 5.3, 6.2,7.0, 7.4 and 8.2row D: 25% PEG 6000 and 30% 2-propanol with buffers pH 4.8, 5.3, 6.2,7.0, 7.4 and 8.2Results: Strong liquid phase separation in all samples, gel phase in 30%2-propanol.Microscopy observationsC = crystalsA = amorphous precipitateL = liquid phase separation, spherical dropletsG = gel, glassy solid irregular particlesN = no phase separations, clear solutionX = experiment failed, discontinued, dried, microbial contamination etc.Recombinant Production

A growing number of recombinant proteins are being developed fortherapeutic and diagnostic applications. However, many of these proteinsmay be difficult or expensive to produce in a functional form and/or inthe required quantities using conventional methods. Conventional methodsinvolve inserting the gene responsible for the production of aparticular protein into host cells such as bacteria, yeast, or mammaliancells, e.g., COS or CHO cells, and then growing the cells in culturemedia. The cultured cells then synthesize the desired protein.Traditional bacteria or yeast systems may be unable to produce manycomplex proteins in a functional form. While mammalian cells canreproduce complex proteins, they are generally difficult and expensiveto grow, and often produce only mg/L quantities of protein. In addition,non-secreted proteins are relatively difficult to purify fromprocaryotic or mammalian cells as they are not secreted into the culturemedium.

In general, the transgenic technology features, a method of making andsecreting a protein which is not normally secreted (a non-secretedprotein). The method includes expressing the protein from a nucleic acidconstruct which includes:

-   -   (a) a promoter, e.g., a mammary epithelial specific promoter,        e.g., a milk protein promoter;    -   (b) a signal sequence which can direct the secretion of a        protein, e.g. a signal sequence from a milk specific protein;    -   (c) optionally, a sequence which encodes a sufficient portion of        the amino terminal coding region of a secreted protein, e.g., a        protein secreted into milk, to allow secretion, e.g., in the        milk of a transgenic mammal, of the non-secreted protein; and    -   (d) a sequence which encodes a non-secreted protein,    -   wherein elements (a), (b), optionally (c), and (d) are        preferably operatively linked in the order recited.

In preferred embodiments: elements a, b, c (if present), and d are fromthe same gene; the elements a, b, c (if present), and d are from two ormore genes.

In preferred embodiments the secretion is into the milk of a transgenicmammal.

In preferred embodiments: the signal sequence is the β-casein signalsequence; the promoter is the β-casein promoter sequence.

In preferred embodiments the non-secreted protein-coding sequence: is ofhuman origin; codes for a truncated, nuclear, or a cytoplasmicpolypeptide; codes for human serum albumin or other desired protein ofinterest.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Milk Specific Promoters

The transcriptional promoters useful in practicing the present inventionare those promoters that are preferentially activated in mammaryepithelial cells, including promoters that control the genes encodingmilk proteins such as caseins, beta lactoglobulin (Clark et al., (1989)Bio/Technology 7: 487-492), whey acid protein (Gorton et al. (1987)Bio/Technology 5: 1183-1187), and lactalbumin (Soulier et al., (1992)FEBS Letts. 297: 3). Casein promoters may be derived from the alpha,beta, gamma or kappa casein genes of any mammalian species; a preferredpromoter is derived from the goat beta casein gene (DiTullio, (1992)Bio/Technology 10:74-77). The milk-specific protein promoter or thepromoters that are specifically activated in mammary tissue may bederived from either cDNA or genomic sequences. Preferably, they aregenomic in origin.

DNA sequence information is available for all of the mammary glandspecific genes listed above, in at least one, and often severalorganisms. See, e.g., Richards et al., J. Biol. Chem. 256, 526-532(1981) (α-lactalbumin rat); Campbell et al., Nucleic Acids Res. 12,8685-8697 (1984) (rat WAP); Jones et al., J. Biol. Chem. 260, 7042-7050(1985) (rat β-casein); Yu-Lee & Rosen, J. Biol. Chem. 258, 10794-10804(1983) (rat γ-casein); Hall, Biochem. J. 242, 735-742 (1987)(α-lactalbumin human); Stewart, Nucleic Acids Res. 12, 389 (1984)(bovine αs1 and κ casein cDNAs); Gorodetsky et al., Gene 66, 87-96(1988) (bovine β casein); Alexander et al., Eur. J. Biochem. 178,395-401 (1988) (bovine κ casein); Brignon et al., FEBS Lett. 188, 48-55(1977) (bovine αS2 casein); Jamieson et al., Gene 61, 85-90 (1987),Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429 (1988), Alexanderet al., Nucleic Acids Res. 17, 6739 (1989) (bovine β lactoglobulin);Vilotte et al., Biochimie 69, 609-620 (1987) (bovine α-lactalbumin). Thestructure and function of the various milk protein genes are reviewed byMercier & Vilotte, J. Dairy Sci. 76, 3079-3098 (1993) (incorporated byreference in its entirety for all purposes). To the extent thatadditional sequence data might be required, sequences flanking theregions already obtained could be readily cloned using the existingsequences as probes. Mammary-gland specific regulatory sequences fromdifferent organisms are likewise obtained by screening libraries fromsuch organisms using known cognate nucleotide sequences, or antibodiesto cognate proteins as probes.

Signal Sequences

Among the signal sequences that are useful in accordance with thisinvention are milk-specific signal sequences or other signal sequenceswhich result in the secretion of eukaryotic or prokaryotic proteins.Preferably, the signal sequence is selected from milk-specific signalsequences, i.e., it is from a gene which encodes a product secreted intomilk. Most preferably, the milk-specific signal sequence is related tothe milk-specific promoter used in the expression system of thisinvention. The size of the signal sequence is not critical for thisinvention. All that is required is that the sequence be of a sufficientsize to effect secretion of the desired recombinant protein, e.g., inthe mammary tissue. For example, signal sequences from genes coding forcaseins, e.g., alpha, beta, gamma or kappa caseins, beta lactoglobulin,whey acid protein, and lactalbumin are useful in the present invention.The preferred signal sequence is the goat β-casein signal sequence.

Signal sequences from other secreted proteins, e.g., proteins secretedby liver cells, kidney cell, or pancreatic cells can also be used.

Transgenic Mammals

The DNA constructs of the protein of interest, in this case human serumalbumin, are introduced into the germ line of a mammal. For example, oneor several copies of the construct may be incorporated into the genomeof a mammalian embryo by standard transgenic techniques.

Any non-human mammal can be usefully employed in this invention. Mammalsare defined herein as all animals, excluding humans, that have mammaryglands and produce milk. Preferably, mammals that produce large volumesof milk and have long lactating periods are preferred. Preferred mammalsare cows, sheep, goats, mice, oxen, camels and pigs. Of course, each ofthese mammals may not be as effective as the others with respect to anygiven expression sequence of this invention. For example, a particularmilk-specific promoter or signal sequence may be more effective in onemammal than in others. However, one of skill in the art may easily makesuch choices by following the teachings of this invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of understanding, it willbe apparent to those skilled in the art that certain changes andmodifications may be practiced. Therefore, the description and examplesshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims.

It should also be noted that while albumin is crystallized with variouscompounds, ethanol and mineral salts including phosphates industrialmethods for crystallization with phosphates are not found in theliterature. Through the preferred embodiments of the current inventionit has now been found that human albumin can be crystallizedadvantageously with phosphate salts by utilizing in full extent theinvented key process parameters and/or conditions of the currentinvention. The invented parameters and some variations thereof arelisted and described above.

Accordingly, it is to be understood that the embodiments of theinvention herein providing for crystallized and purified human albuminare merely illustrative of the application of the principles of theinvention. It will be evident from the foregoing description thatchanges in the form, methods of use, and applications of the elements ofthe disclosed may be resorted to without departing from the spirit ofthe invention, or the scope of the appended claims.

PRIOR ART CITATIONS INCORPORATED BY REFERENCE

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1. A crystalline human albumin product comprising at least a portion ofthe material resulting from a process of: a) concentrating an albumincontaining fluid until said fluid has at least 15 grams of albumin perliter of solution; b) adding a first phosphate mixture to said albumincontaining fluid until the concentration of said phosphate mixture is inthe range of 2.4-2.6 molar; c) providing a first filtering of saidalbumin containing solution so as to form a resultant crystallizingbatch solution to remove impurities; d) cooling the resultant filtrateof said crystallizing batch solution to a temperature of at most 15° C.;e) allowing the human albumin in said crystallizing batch solution tocrystallize; f) adding more of said first phosphate mixture to saidcrystallizing batch solution sufficient to achieve a concentration of atmost 3.0 molar; g) making a first separation of albumin crystals fromany remaining fluid; h) suspending the albumin crystals from said firstseparation of albumin crystals in a second phosphate mixture whereinsaid second phosphate mixture has a concentration of 2.7-3.0 M; i)heating the crystal suspension from said first separation of albumincrystals up to temperature in the range of 40-50° C. in order todissolve the crystals, j) providing a second filtering to the dissolvedcrystal suspension from said first separation of albumin crystals; k)cooling the resultant dissolved crystal suspension from said firstseparation of albumin crystals to a temperature of at most 15° C.; andl) allowing albumin crystals to form the resultant cooled crystalsuspension; wherein the application of these specific steps allows thepurification and crystallization of human albumin from a given humanalbumin containing feedstream.
 2. The process of claim 1, wherein saidfirst phosphate mixture is comprised of a sodium phosphate salt.
 3. Theprocess of claim 1, wherein said first phosphate mixture is a potassiumphosphate salt.
 4. The process of claim 1, wherein said first phosphatemixture is comprised of both a sodium and a potassium salt.
 5. Theprocess of claim 1, wherein said albumin containing fluid has aconcentration of albumin in one liter of solution in a range of 15-50grams.
 6. The process of claim 1, wherein the temperature of said firstphosphate mixture at the time of addition is in the range of 20-30° C.7. The process of claim 1, wherein said first phosphate mixture at thetime of addition has a pH in the range of pH 6.0-6.7.
 8. The process ofclaim 1, wherein said filtrate collected after filtering saidcrystallizing batch is cooled to a temperature of at most 10° C.
 9. Theprocess of claim 1, wherein said crystallizing batch solution is allowedto crystallize for at most 12 hours.
 10. The process of claim 1, whereinsaid crystallizing batch solution is allowed to crystallize for at most24 hours.
 11. The process of claim 1, wherein said crystallizing batchsolution is allowed to crystallize for at least 24 hours
 12. The processof claim 1, wherein said first separation of albumin crystals isaccomplished by filtration.
 13. The process of claim 1, wherein saidfirst separation of albumin crystals is accomplished by centrifugation.14. The process of claim 1, wherein said first separation of albumincrystals is accomplished by gravity.
 15. The process of claim 1, whereinsaid first separation of albumin crystals is accomplished by drying. 16.The process of claim 1, wherein said second phosphate mixture iscomprised of a sodium phosphate salt.
 17. The process of claim 1,wherein said second phosphate mixture is a potassium phosphate salt. 18.The process of claim 1, wherein said second phosphate mixture iscomprised of both a sodium and a potassium salt.
 19. The process ofclaim 1, wherein said dissolved crystal suspension from said firstseparation of albumin crystals is cooled to a temperature of at most 10°C.
 20. The process of claim 19, wherein the albumin crystalsprecipitating out of the dissolved crystal suspension are dissolved backinto solution again and re-crystallized at least once.
 21. The processof claim 1, wherein said albumin containing fluid is previouslyclarified to remove impurities not in solution.
 22. The process of claim1, wherein said feedstream is milk or other bodily fluid from atransgenic mammal.
 23. The process of claim 22, wherein milk from atransgenic mammal is clarified to remove impurities and some milkproteins.
 24. The process of claim 22, wherein the level of purity in agiven feedstream is at least 10%, that is, wherein human albuminconstitutes at least 10% of the total protein of a given solution. 25.The process according to claim 1, wherein the pH of said resultantcrystallizing batch solution is 5.6-7.8.
 26. The process according toclaim 1, wherein the pH of said resultant crystallizing batch solutionis 6.0-6.5.
 27. The process according to claim 1, wherein the pH of saidresultant crystallizing batch solution is 7.0-7.8.
 28. The processaccording to claim 1, wherein said the concentration of said firstphosphate mixture has a concentration in the range of 2.2-3.0 M.
 29. Theprocess of claim 1, wherein said albumin containing fluid has an albuminconcentration in a range of 2-400 grams per liter of solution.
 30. Theprocess of claim 1, wherein said albumin containing fluid has an albuminconcentration in a range of 3-300 grams per liter of solution.
 31. Theprocess of claim 1, wherein said albumin containing fluid has an albuminconcentration in a range of 3-100 grams per liter of solution.
 32. Theprocess of claim 1, wherein said albumin containing fluid has an albuminconcentration in a range of 3-40 grams per liter of solution.
 33. Theprocess of claim 1, wherein said albumin containing fluid has an albuminconcentration in a range of 2-10 grams per liter of solution.
 34. Theprocess of claim 1, wherein said crystalline human albumin product isutilized as an excipient in pharmaceutical preparations.
 35. The processof claim 1, wherein said crystalline human albumin product is utilizedas a therapeutic agent in a pharmaceutical composition.
 36. The processof claim 1, wherein said feedstream is a culture supernatant or bodilyfluid derived from a host which expresses recombinant human albumin. 37.The process of claim 36, wherein said host is a mammalian cell culture.38. The process of claim 36, wherein said host is a yeast cell culture.39. The process of claim 36, wherein said host is an insect cellculture.
 40. The process of claim 36, wherein said host is a prokaryoticcell culture.
 41. The process of claim 1, wherein said crystalline humanalbumin product is utilized to treat a medical condition.
 42. Theprocess of claim 41, wherein said medical condition is selected from thegroup consisting of: a) Edema; b) Hypovolemia; c) Hypoalbuminemia; d)Adult Respiratory Distress Syndrome (ARDS); e) Nephrosis; f) HemolyticDisease of the Newborn (HDN); g) Severe burn; h) Hypoproteinemia; and,i) Acute pancreatitis,
 43. The process of claim 1, wherein saidcrystalline human albumin product is utilized during cardiopulmonarybypass surgery. 44-86. (canceled)
 87. A human albumin product comprisingat least a portion of the material resulting from a process of: a)concentrating an albumin containing fluid until said fluid has at least15 grams of albumin per liter of solution; b) adding a first phosphatemixture to said albumin containing fluid until the concentration of saidphosphate mixture is in the range of 2.6-3.0 molar and the pH is in therange of 6.1-6.3; c) cooling the resultant filtrate of saidcrystallizing batch solution to a temperature of at most 15° C.; d)allowing the human albumin in said crystallizing batch solution tocrystallize; and e) separating the albumin crystals from any remainingfluid; wherein the application of these specific steps allows thepurification and crystallization of human albumin from a given humanalbumin containing feedstream.
 88. The process of claim 87, wherein saidfirst phosphate mixture is comprised of a sodium phosphate salt.
 89. Theprocess of claim 87, wherein said first phosphate mixture is a potassiumphosphate salt.
 90. The process of claim 87, wherein said firstphosphate mixture is comprised of both a sodium and a potassium salt.91. The process of claim 87, wherein said albumin containing fluid has aconcentration of albumin in one liter of solution in a range of 15-50grams.
 92. The process of claim 87, wherein the temperature of saidfirst phosphate mixture at the time of addition is in the range of20-30° C.
 93. The process of claim 87, wherein said crystallizing batchsolution is allowed to crystallize for at most 12 hours.
 94. The processof claim 87, wherein said crystallizing batch solution is allowed tocrystallize for at most 24 hours.
 95. The process of claim 87, whereinsaid crystallizing batch solution is allowed to crystallize for at least24 hours
 96. The process of claim 87, wherein said first separation ofalbumin crystals is accomplished by filtration.
 97. The process of claim87, wherein said first separation of albumin crystals is accomplished bycentrifugation.
 98. The process of claim 87, wherein said firstseparation of albumin crystals is accomplished by gravity.
 99. Theprocess of claim 87, wherein said first separation of albumin crystalsis accomplished by drying.
 100. The process of claim 87, wherein thealbumin crystals precipitating out of the dissolved crystal suspensionare dissolved back into solution again and re-crystallized at leastonce.
 101. The process of claim 87, wherein said human albumincontaining fluid from said feedstream is previously clarified to removeimpurities not in solution.
 102. The process of claim 87, wherein saidfeedstream is milk or other bodily fluid from a transgenic mammal. 103.The process of claim 102, wherein milk from a transgenic mammal isclarified to remove impurities and some milk proteins.
 104. The processof claim 102, wherein the level of purity in a given feedstream is atleast 10%, that is, wherein human albumin constitutes at least 10% ofthe total protein of a given solution.
 105. The process of claim 87,wherein said albumin containing fluid has an albumin concentration in arange of 2-400 grams per liter of solution.
 106. The process of claim87, wherein said albumin containing fluid has an albumin concentrationin a range of 3-300 grams per liter of solution.
 107. The process ofclaim 87, wherein said albumin containing fluid has an albuminconcentration in a range of 3-100 grams per liter of solution.
 108. Theprocess of claim 87, wherein said albumin containing fluid has analbumin concentration in a range of 3-40 grams per liter of solution.109. The process of claim 87, wherein said albumin containing fluid hasan albumin concentration in a range of 2-10 grams per liter of solution.110. The process of claim 87, wherein said crystalline human albuminproduct is utilized as an excipient in pharmaceutical preparations. 111.The process of claim 87, wherein said crystalline human albumin productis utilized as a therapeutic agent in a pharmaceutical composition. 112.The process of claim 87, wherein said feedstream is or is derived from aculture supernatant or bodily fluid from a host which expressesrecombinant human albumin.
 113. The process of claim 112, wherein saidhost is a mammalian cell culture.
 114. The process of claim 112, whereinsaid host is a transgenic mammal.
 115. The process of claim 112, whereinsaid host is a transgenic avian.
 116. The process of claim 112, whereinsaid host is a transgenic plant.
 117. The process of claim 112, whereinsaid host is a yeast cell culture.
 118. The process of claim 112,wherein said host is an insect cell culture.
 119. The process of claim112, wherein said host is a prokaryotic cell culture.
 120. The processof claim 87, wherein said crystalline human albumin product is utilizedto treat a medical condition.
 121. The process of claim 120, whereinsaid medical condition is selected from the group consisting of: a)Edema; b) Hypovolemia; c) Hypoalbuminemia; d) Adult Respiratory DistressSyndrome (ARDS); e) Nephrosis; f) Hemolytic Disease of the Newborn(HDN); g) Severe burn; h) Hypoproteinemia; and, i) Acute pancreatitis,122. The process of claim 87, wherein said crystalline human albuminproduct is utilized during cardiopulmonary bypass surgery.
 123. Theprocess of claim 87, in which the albumin crystals are further purifiedby one or more recrystallization steps comprising: a) suspending thealbumin crystals from said first separation of albumin crystals in asecond phosphate mixture wherein said second phosphate mixture has aconcentration of 2.7-3.0 M and a pH in the range of 6.1-6.3; b) heatingthe crystal suspension from said first separation of albumin crystals upto temperature in the range of 40-50° C. in order to dissolve thecrystals, c) providing a second filtering to the dissolved crystalsuspension from said first separation of albumin crystals; d) coolingthe resultant dissolved crystal suspension from said first separation ofalbumin crystals to a temperature of at most 15° C.; e) allowing albumincrystals to form from the resultant cooled crystal suspension; and f)separating the albumin crystals from any remaining fluid; wherein theapplication of these specific steps allows the purification andcrystallization of human albumin from a given feedstream.
 124. Theprocess of claim 123, wherein said second phosphate mixture is comprisedof a sodium phosphate salt.
 125. The process of claim 123, wherein saidsecond phosphate mixture is a potassium phosphate salt.
 126. The processof claim 123, wherein said second phosphate mixture is comprised of botha sodium and a potassium salt.
 127. A human albumin product comprisingat least a portion of the material resulting from a process of: a)concentrating an albumin containing fluid until said fluid has at least15 grams of albumin per liter of solution; b) adding a first phosphatemixture to said albumin containing fluid until the concentration of saidphosphate mixture is at most 2.6 molar and the pH is in the range of6.1-6.3; c) providing a first filtering of said albumin containingsolution so as to form a resultant crystallizing batch solution toremove impurities; d) cooling the resultant filtrate of saidcrystallizing batch solution to a temperature of at most 15° C.; e)adding more of said first phosphate mixture to said crystallizing batchsolution sufficient to achieve a concentration of at most 3.0 molar; f)allowing the human albumin in said crystallizing batch solution tocrystallize; and g) separating the albumin crystals from any remainingfluid; wherein the application of these specific steps allows thepurification and crystallization of human albumin from a givenfeedstream.
 128. The process of claim 127, wherein said first phosphatemixture is comprised of a sodium phosphate salt.
 129. The process ofclaim 127, wherein said first phosphate mixture is a potassium phosphatesalt.
 130. The process of claim 127, wherein said first phosphatemixture is comprised of both a sodium and a potassium salt.
 131. Theprocess of claim 127, wherein said albumin containing fluid has aconcentration of albumin in one liter of solution in a range of 15-50grams.
 132. The process of claim 127, wherein the temperature of saidfirst phosphate mixture at the time of addition is in the range of20-30° C.
 133. The process of claim 127, wherein said crystallizingbatch solution is allowed to crystallize for at most 12 hours.
 134. Theprocess of claim 127, wherein said crystallizing batch solution isallowed to crystallize for at most 24 hours.
 135. The process of claim127, wherein said crystallizing batch solution is allowed to crystallizefor at least 24 hours.
 136. The process of claim 127, wherein said firstseparation of albumin crystals is accomplished by filtration.
 137. Theprocess of claim 127, wherein said first separation of albumin crystalsis accomplished by centrifugation.
 138. The process of claim 127,wherein said first separation of albumin crystals is accomplished bygravity.
 139. The process of claim 127, wherein said first separation ofalbumin crystals is accomplished by drying.
 140. The process of claim127, wherein the albumin crystals precipitating out of the dissolvedcrystal suspension are dissolved back into solution again andre-crystallized at least once.
 141. The process of claim 127, whereinsaid human albumin containing fluid from said feedstream is previouslyclarified to remove impurities not in solution.
 142. The process ofclaim 127, wherein said feedstream is milk or other bodily fluid from atransgenic mammal.
 143. The process of claim 102, wherein milk from atransgenic mammal is clarified to remove impurities and some milkproteins.
 144. The process of claim 102, wherein the level of purity ina given feedstream is at least 10%, that is, wherein human albuminconstitutes at least 10% of the total protein of a given solution. 145.The process of claim 127, wherein said albumin containing fluid has analbumin concentration in a range of 2-400 grams per liter of solution.146. The process of claim 127, wherein said albumin containing fluid hasan albumin concentration in a range of 3-300 grams per liter ofsolution.
 147. The process of claim 127, wherein said albumin containingfluid has an albumin concentration in a range of 3-100 grams per literof solution.
 148. The process of claim 127, wherein said albumincontaining fluid has an albumin concentration in a range of 3-40 gramsper liter of solution.
 149. The process of claim 127, wherein saidalbumin containing fluid has an albumin concentration in a range of 2-10grams per liter of solution.
 150. The process of claim 127, wherein saidcrystalline human albumin product is utilized as an excipient inpharmaceutical preparations.
 151. The process of claim 127, wherein saidcrystalline human albumin product is utilized as an therapeutic agent ina pharmaceutical composition.
 152. The process of claim 127, whereinsaid feedstream is a culture supernatant or bodily fluid from a hostwhich expresses recombinant human albumin.
 153. The process of claim152, wherein said host is a mammalian cell culture.
 154. The process ofclaim 152, wherein said host is a transgenic mammal.
 155. The process ofclaim 152, wherein said host is a transgenic avian.
 156. The process ofclaim 152, wherein said host is a transgenic plant.
 157. The process ofclaim 152, wherein said host is a yeast cell culture.
 158. The processof claim 152, wherein said host is an insect cell culture.
 159. Theprocess of claim 152, wherein said host is a prokaryotic cell culture.160. The process of claim 127, wherein said crystalline human albuminproduct is utilized to treat a medical condition.
 161. The process ofclaim 160, wherein said medical condition is selected from the groupconsisting of: a) Edema; b) Hypovolemia; c) Hypoalbuminemia; d) AdultRespiratory Distress Syndrome (ARDS); e) Nephrosis; f) Hemolytic Diseaseof the Newborn (HDN); g) Severe burn; h) Hypoproteinemia; and, i) Acutepancreatitis,
 162. The process of claim 127, wherein said crystallinehuman albumin product is utilized during cardiopulmonary bypass surgery.163. The process of claim 127, in which the albumin crystals are furtherpurified by one or more recrystallization steps comprising: g)suspending the albumin crystals from said first separation of albumincrystals in a second phosphate mixture wherein said second phosphatemixture has a concentration of 2.7-3.0 M and a pH in the range of6.1-6.3; h) heating the crystal suspension from said first separation ofalbumin crystals up to temperature in the range of 40-50° C. in order todissolve the crystals, i) providing a second filtering to the dissolvedcrystal suspension from said first separation of albumin crystals; j)cooling the resultant dissolved crystal suspension from said firstseparation of albumin crystals to a temperature of at most 15° C.; k)allowing albumin crystals to form from the resultant cooled crystalsuspension; and l) separating the albumin crystals from any remainingfluid; wherein the application of these specific steps allows thepurification and crystallization of human albumin from a givenfeedstream.
 164. The process of claim 163, wherein said second phosphatemixture is comprised of a sodium phosphate salt.
 165. The process ofclaim 163, wherein said second phosphate mixture is a potassiumphosphate salt.
 166. The process of claim 163, wherein said secondphosphate mixture is comprised of both a sodium and a potassium salt.167. The process of claim 1, wherein said human albumin containingfeedstream contains a compound bound to a dissolved human serum albuminproduct, said compound being selected from the group including: a)Caprylate; b) A fatty acid; and c) A long chain alcohol.
 168. Theprocess of claim 1, wherein said human albumin product is purified to aconcentrate said concentrate being in one of a variety of phases saidcondition being selected from the group consisting of: a) Crystalline;b) gel; c) precipitant; and d) droplet
 169. The process of claim 87,wherein said human albumin containing feedstream contains a compoundbound to a dissolved human serum albumin product, said compound beingselected from the group including: a) Caprylate; b) A fatty acid; and c)A long chain alcohol.
 170. The process of claim 87, wherein said humanalbumin product is purified to a concentrate said concentrate being inone of a variety of phases said condition being selected from the groupconsisting of: a) Crystalline; b) gel; c) precipitant; and d) droplet171. The process of claim 127, wherein said human albumin containingfeedstream contains a compound bound to a dissolved human serum albuminproduct, said compound being selected from the group including: a)Caprylate; b) A fatty acid; and c) A long chain alcohol.
 172. Theprocess of claim 127, wherein said human albumin product is purified toa concentrate said concentrate being in one of a variety of phases saidcondition being selected from the group consisting of: a) Crystalline;b) gel; c) precipitant; and d) droplet 173-183. (canceled)